Non-lactone carbocyclic and heterocyclic antagonists and agonists of bacterial quorum sensing

ABSTRACT

Compounds which modulate quorum sensing in quorum sensing bacteria. Compounds of the invention inhibit quorum sensing and/or activate quorum sensing in various bacteria. Compounds that inhibit quorum sensing are particularly useful for inhibition of detrimental bacterial biofilm formation. Compounds that activate quorum sensing are particularly useful for promoting growth and biofilm formation of beneficial bacterial.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application61/222,075, filed Jun. 30, 2009, which is incorporated by referenceherein in its entirety.

STATEMENT REGARDING GOVERNMENT FUNDING FOR RESEARCH AND DEVELOPMENT

This invention was made with government support under AI063326 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

Quorum sensing (QS) is a process by which bacteria assess theirpopulation density through a language of low molecular weight signallingmolecules (autoinducers). Gram-negative bacteria commonly use N-acylatedhomoserine lactones (AHLs) as their primary autoinducers and theirrespective receptors (R proteins) for QS. Assessing population densityallows for the modulation of gene expression levels required for groupbehaviour. Genes regulated by QS in Pseudomonas aeruginosa includevirulence factor production and biofilm production. [Geske, G. D.;O'Neill, J. C.; Miller, D. M.; Mattmann, M. E.; Blackwell, H. E.,Modulation of Bacterial Quorum Sensing: Systematic Evaluation ofN-Acylated Homoserine Lactones in Multiple Species and New Insights intoTheir Mechanism of Action. J. Am. Chem. Soc. 2007, 129, 13613-13625.]

At high cell densities, bacteria use this chemical signaling process toswitch from a nomadic existence to that of multicellular community. Thislifestyle switch is significant, as numerous pathogenic bacteria usequorum sensing to turn on virulence pathways and form drug-imperviouscommunities called biofilms that are the basis of myriad chronicinfections. Over 80% of bacterial infections in humans involve theformation of biofilms, as exemplified in lung infections by Pseudomonasaeruginosa, which is the primary cause of morbidity in cystic fibrosispatients. The treatment of infections by pathogens that form biofilmscosts over $1 billion/year in the US alone. Biofilms are denseextracellular polymeric matrices in which the bacteria embed themselves.Biofilms allow bacteria to create a microenviroment that attaches thebacteria to the host surface and which contains excreted enzymes andother factors allowing the bacteria to evade host immune responsesincluding antibodies and cellular immune responses. Such biofilms canalso exclude antibiotics. Further, biofilms can be extremely resistantto removal and disinfection. For individuals suffering from cysticfibrosis, the formation of biofilms by P. aeruginosa is eventuallyfatal. Other bacteria also respond to quorum sensing signals byproducing biofilms. Biofilms are inherent in dental plaques, and arefound on surgical instruments, food processing and agriculture equipmentand water treatment and power generating machinery and equipment.

Gram-negative bacteria represent numerous relevant pathogens usingquorum-sensing pathways. Besides P. aeruginosa, other quorum sensingbacteria include: Aeromonas hydrophila, A. salmonicida, Agrobacteriumtumefaciens, Burkholderia cepacia, Chromobacterium violaceum,Enterobacter agglomeran, Erwinia carotovora, E. chrysanthemi,Escherichia coli, Nitrosomas europaea, Obesumbacterium proteus, Pantoeastewartii, Pseudomonas aureofaciens, P. syringae, Ralstoniasolanacearum, Rhisobium etli, R. leguminosarum, Rhodobacter sphaeroides,Serratia liguefaciens, S. marcescens, Vibrio anguillarum, V. fischeri,V. cholerae, Xenorhabdus nematophilus, Yersinia enterocolitica, Y.pestis, Y. pseudotuberculosis, Y. medievalis, and Y. ruckeri. Studies onthe above listed bacteria indicate that, while the Al is generally anAHL compound, the genes affected as well as the phenotypes resultingfrom induction of the promoter differ according to the particular lifecycle of each bacterium. Further, quorum sensing stimulation typicallyresults in altered expression of multiple genes.

P. aeruginosa is an opportunistic pathogen that causes severe, oftenfatal, infections in burn victims and cystic fibrosis patients and istherefore of direct and profound biomedical importance. P. aeruginosauses 3-oxo-dodecanoyal homoserine lactone (OdDHL) as its autoinducer(Compound A):

While successful modifications to the acyl tail region of autoinducershave been made, modifications to the AHL head group have met limitedsuccess. Modifications to the head group are important because thelactone ring is prone to hydrolysis at pH 7 and higher. [Glansdorp, F.G.; Thomas, G. L.; Lee, J. K.; Dutton, J. M.; Salmond, G. P. C.; Welch,M.; Spring, D. R., Synthesis and stability of small molecule probes forPseudomonas aeruginosa quorum sensing modulation. Org. Biomol. Chem.2004, 2, 3329-3336.] This work relates to non-homoserine lactone-basedautoinducer analogs for QS modulation and provides a betterunderstanding of the structural and electronic requirements of theautoinducer's head group. Certain of the compounds of this invention aredesigned as autoinducer analogs for QS modulation in P. aeruginosa.

Previous work in the field of P. aeruginosa QS modulators showed thatmany active non-lactone structures are highly conjugated and retain someform of the acyl chain, suggesting that a region of hydrophobicity inthe acyl tail region is critical. [Muh, U.; Schuster, M.; Heim, R.;Singh, A.; Olson, E.; Greenberg, E. P., Novel Pseudomonas aeruginosaQuorum-Sensing Inhibitors Identified in an Ultra-High-Throughput Screen.Antimicrob. Agents Chemother. 2006, 50, 3674-3679; Muh, U.; Hare, B. L.;Duerkop, B. A.; Schuster, M.; Hanzelka, B. L.; Heim, R.; Olson, E. R.;Greenberg, E. P., A Structurally Unrelated Mimic of a Pseudomonasaeruginosa acyl-homoserine lactone quorum sensing signal. Proc. Natl.Acad. Sci. U.S.A. 2006, 103, 16948-16952; Lee, L. Y. W.; Hupfield, T.;Nicholson, R. L.; Hodgkinson, J. T.; Su, X.; Thomas, G. L.; Salmond, P.C.; Welch, M.; Spring, D. R., 2-Methoxycyclopentyl analogues of aPseudomonas aeruginosa quorum sensing modulator. Molecular BioSystems2008, 4, 505-507; Eberhard, A.; Widrig, C. A.; MaBath, P.; Schineller,J. B., Analogs of the autoinducer of bioluminescence in Vibrio fischeri.Arch. Microbiol. 1986, 146, 35-40; Rasmussen, T. B.; Givskov, M., Quorumsensing inhibitors: a bargain of effects. Microbiology 2006, 152,895-904; Hjelmgaard, T.; Persson, T.; Rasmussen, T. B.; Givskov, M.;Nielsen, J., Synthesis of Furanone-based natural product analogues withquorum sensing antagonist activity. Bioorg. Med. Chem. 2003, 11,3261-3271; Smith, K. M.; Bu, Y.; Suga, H., Induction and Inhibition ofPseudomonas aeruginosa quorum sensing by synthetic autoinducer analogs.Chem. Biol. 2003, 10, 81-89; Schaefer, A. L.; Hanzelka, B. L.; Eberhard,A.; Greenberg, E. P., Quorum sensing in Vibrio fischeri: Probingautoinducer-LuxR interactions with autoinducer analogs. J. Bacteriol.1996, 178, 2897-2901; Passador, L.; Tucker, K. D.; Guertin, K. R.;Journet, M. P.; Kende, A. S.; Iglewski, B. H., Functional analysis ofthe Pseudomonas aeruginosa Autoinducer PAI. J. Bacteriol. 1996, 178,5995-6000; Smith, K. M.; Bu, Y.; Suga, H., Library Screening forSynthetic Agonists and Antagonists of a Pseudomonas aeruginosaautoinducer. Chem. Biol. 2003, 10, 563-571; Ishida, T.; Ikeda, T.;Takiguchi, N.; Kuroda, A.; Ohtake, H.; Kato, J., Inhibition of quorumsensing in Pseudomonas aeruginosa by N-acyl cyclopentylamides. Appl.Environ. Microbiol. 2007, 73, 3183-3188; Fletcher, M. P.; Diggle, S. P.;Crusz, S. A.; Chhabra, S. R.; Camara, M.; Williams, P., A dual biosensorfor 2-alkyl-4-quinolone quorum sensing signal molecules. Environ.Microbiol. 2007, 9, 2683-2693; Kim, C.; Kim, J.; Park, H. Y.; Park, H.J.; Lee, J. H.; Kim, C. K.; Yoon, J., Furanone derivatives as quorumsensing antagonists of Pseudomonas aeruginosa. Appl. Microbiol.Biotechnol. 2008, 80, 37-47; Estephane, J.; Dauvergne, J.; Soulere, L.;Reverchon, S.; Queneau, Y.; Doutheau, A., N-Acyl-3-amino-5H-furanonederivatives as new inhibitors of LuxR-dependent quorum sensing:Synthesis, biological evaluation and binding mode study. Bioorg. Med.Chem. Lett. 2008, 18, 4321-4324.]

Furthermore, a close examination of the crystal structure of theN-terminal domain of LasR reveals a hydrogen bond between the 3-oxocarbonyl in the acyl tail of OdDHL and a water molecule present in theLasR binding site [Bottomley, M. J.; Muraglia, E.; Bazzo, R.; Carfi, A.,Molecular insights into quorum sensing in the human pathogen Pseudomonasaeruginosa from the structure of the virulence regulator LasR bound toits autoinducer. J. Biol. Chem. 2007, 282, 13592-13600.]

Published US application US2006/0178430, published Aug. 10, 2006 andInternational published application WO 2008/116029, published Sep. 25,2008 relate to quorum sensing compounds and their uses. These documentsare incorporated by reference in their entirety herein for theirdescription of the state of the art and for additional methods ofsynthesis, methods of testing, and methods of application of quorumsensing compounds.

Janssens, J. C. A. et al. (2007) Applied Environ. Microbiol. 73 (2)535-544 reports that certain N-acyl homoserine lactones includingcertain thiolactones are strong activators of SdiA, the Salmonellaenterica Serovar Typhimurium LuxR homologues.

Published PCT application WO2002/052949 relates to the use ofautoinducer compounds as additives to animal feeds for improving animalperformance.

SUMMARY OF THE INVENTION

The invention provides a compound of formula I:A-[Z]_(n)-L1-[Y]_(q)—W—[V]_(m)-L2-HGor a pharmaceutically acceptable salt or ester thereofwhere:W is —NH— or

Y is —CO—, —CO—CH₂—CO—, —NH—CO—, —CO—CH₂—C(Y1)-, —SO₂—, where Y1 is —OH,—SH, —NH₂ or —F;q is 1 or 0 to indicate the presence or absence, respectively of Y;L1 and L2, independently, are —[CH₂]_(p1)— and —[CH₂]_(p2)—, where p1and p2, independently, are 0 or integers ranging from 1-10 and one ormore of the carbons of L1 or L2 can be substituted with one or twonon-hydrogen substituents;V is

where R_(N) is an alkyl group having 1-3 carbon atoms;m is 1 or 0 to indicate, respectively, the presence or absence of the Vgroup;Z is —CO—, —O—CO—, —CO—O—, —NH—CO—, —CO—NH—, —NH—CO—NH—, —O—, —S—, or—NH₂—, n is 1 or 0 to indicate, respectively, the presence of absence ofthe Z group;A is an aryl or heteroaryl group having one or two 5- or 6-member ringswith 1-3 heteroatoms in a ring, a C₅-C₈ cycloalkyl group, a C₅-C₈cycloalkenyl group, a heterocyclic group having one or two 5 to 8-memberrings with 1-3 heteroatoms in a ring, a branched or unbranched C₁-C₁₂acyclic aliphatic group, all of which groups can have one or morenon-hydrogen substituents selected from the group consisting of halogen,nitro, hydroxyl, nitrile, azide, —R, —OR, —COOR, —OCOR, —COR, —OCOOR,—CON(R)₂, —OCON(R)₂, —N(R)₂, —SR, —SO₂R, —SOR, and —SO₂N(R)₂, whereineach R is independently selected from the group consisting of hydrogen,an amine group, a substituted or unsubstituted unbranched C₁-C₁₂ acyclicaliphatic group, a substituted or unsubstituted branched C₁-C₁₂ acyclicaliphatic group, a substituted or unsubstituted C₃-C₈ cycloalkyl group,a substituted or unsubstituted C₃-C₈ cycloalkenyl group, a fluorinatedC₁-C₁₂ alkyl group, a substituted or unsubstituted aryl group, asubstituted or unsubstituted heterocyclic group, and a protecting group;additionally, two R groups in the same substituent, optionally form a4-8 member ring; andHG is a head group selected from an aryl or heteroaryl group having oneor two 5- or 6-member rings with 1-3 heteroatoms in a ring; a C₅-C₈cycloalkyl group; a C₅-C₈ cycloalkenyl group; a heterocyclic grouphaving one or two 5 to 8-member rings with 1-3 heteroatoms in a ring; analkyl group having 1-3 carbon atoms substituted with two aryl orheteroaryl groups; a cyclic lactone, lactam, thiolactone or ketone grouphaving a 4-8 member ring, or an ester group R_(E)—O—CO—, where R_(E) isan optionally substituted alkyl group having 1-6 carbon atoms; all ofwhich groups can have one or more non-hydrogen substituents selectedfrom the group consisting of halogen, nitro, hydroxyl, nitrile, azide,—R, —OR, —COOR, —OCOR, —COR, —OCOOR, —CON(R)₂, —OCON(R)₂, —N(R)₂, —SR,—SO₂R, —SOR, and —SO₂N(R)₂, wherein each R is independently selectedfrom the group consisting of hydrogen, an amine group, a substituted orunsubstituted unbranched C₁-C₁₂ acyclic aliphatic group, a substitutedor unsubstituted branched C₁-C₁₂ acyclic aliphatic group, a substitutedor unsubstituted C₃-C₈ cycloalkyl group, a substituted or unsubstitutedC₃-C₈ cycloalkenyl group, a fluorinated C₁-C₁₂ alkyl group, asubstituted or unsubstituted aryl group, a substituted or unsubstitutedheterocyclic group, and a protecting group; additionally, two R groupson the same substituent optionally form a 4-8 member ring.

In specific embodiments, n is 0. In specific embodiments, m is 0. Inspecific embodiments, n is 0 and m is 0. In specific embodiments, n is 0and q is 1. In specific embodiments, n is 0, m is 0 and q is 1. Inspecific embodiments, n is 0, m is 1 and q is 1. In specificembodiments, n is 1, m is 1 and q is 1.

In a specific embodiment, W is —NH—.

In an embodiment, HG is a group having formula:

where r is an integer ranging from 1-4, G is —O—, —S—, —NH— or —CH₂—; R′is hydrogen or a 1-6 carbon aliphatic group, particularly an alkylgroup, and X represents optional substitution with one or morenon-hydrogen substituents on one or more ring carbons. In specificembodiments, r is 1 or 2, the ring is unsubstituted and R′ is H.

In a specific embodiment, G is —S—. In a specific embodiment, G is —S—and r is 1. In a specific embodiment, G is —S—, r is 1 and R′ is analkyl group. In a specific embodiment, G is —S—, r is 1 and R′ is analkyl group. X represents 1, or 2 substituents on the ring.

In an embodiment, HG is a group other than a ketone, lactone, or lactamgroup, when W is —NH—.

In an embodiment, HG is selected from an optionally substituted phenyl,naphthyl, cyclohexyl, cyclohexenyl, cyclopentyl, pyridyl, piperidyl,furyl, thienyl, pyrroyl, or

where r is an integer ranging from 1-4, R′ is hydrogen or a 1-6 carbonaliphatic group, particularly an alkyl group, and X represents optionalsubstitution with one or more non-hydrogen substituents on one or morering carbons. In specific embodiments, r is 1, the ring is unsubstitutedand R′ is H. In specific embodiments, r is 1, and R′ is an alkyl group,particularly a methyl group. In specific embodiments, r is 1, R′ is analkyl group, particularly a methyl group and X represents 1, or 2substituents on the ring.

In specific embodiments, when HG is an unsubstituted thiolactone ring(where G is S and all X and R′ are hydrogen) and W is —NH—, A is a groupother than an unsubstituted alkyl group or a halogenated alkyl group. Inspecific embodiments, when HG is an unsubstituted thiolactone ring(where G is S and all X and R′ are hydrogen); W is —NH—, q is 1 and Y is—COCH₂—CO—, A is a group other than an unsubstituted alkyl group or ahalogenated alkyl group. In specific embodiments, when HG is anunsubstituted thiolactone ring (where G is S and all X and R′ arehydrogen); W is —NH—; m, n, p1 and p2 are all 0; q is 1 and Y is—CO—CH₂—CO—, A is a group other than an unsubstituted alkyl group or ahalogenated alkyl group.

In specific embodiments, HG is a group as illustrated in FIG. 1-1, or1-2, where X, X1 and X2, represent optional substitution with one ormore non-hydrogen substituents on one or more ring carbons. In theseFigures X, X1 and X2 represents optional substitution with one or morenon-hydrogen substituents on one or more ring carbons, RA is H or analkyl group, particularly one having 1-3 carbon atoms. In more specificembodiments, HG is selected from groups HG1, HG4, HG7, HG8, HG10, HG11,or HG12. In other specific embodiments, HG is selected from groups HG2,HG3, HG14, HG15, HG17, HG18 or HG21. In specific embodiments, HG is agroup of any of FIG. 2-1, 2-2, or 2-3. In these Figures X, X1-X5represents optional substitution with one or more non-hydrogensubstituents on one or more ring carbons and R′ is an alkyl group having1-6 or 1-3 carbon atoms.

In specific embodiments HG is an ester group R_(E)—O—CO—, where R_(E) isan unsubstituted alkyl group having 1-6 carbon atoms; an alkyl groupsubstituted with one or more halogens, particularly fluorines; a phenylgroup or optionally substituted phenyl group, particular a phenyl groupsubstituted with one or more halogens, particularly fluorine, one ormore nitro groups, one or more alkoxy groups (including 1C-3C alkoxygroups), or one or more trifluoromethyl groups. In specific embodiments,R_(E) is methyl, ethyl, propyl or butyl groups. In more specificembodiments, R_(E) is a methyl or ethyl group. In specific embodimentswhen HG is an ester group L2 is —CH(CH₃)—.

In specific embodiments, HG is a group as illustrated in FIG. 1-1, 1-2,2-1, 2-2, or 2-3, and A is a branched or unbranched aliphatic grouphaving 1-12 carbon atoms and more specifically is an alkyl or alkenylgroup having 1-12 carbon atoms. In specific embodiments HG is a group asillustrated in FIG. 2-1, 2-2, or 2-3.

In specific embodiments, A is a group as in FIG. 3, where X representsoptional substitution with one or more non-hydrogen substituents on oneor more ring carbons or on a specific ring carbon, R′ is an alkyl group,particularly one having 1-6 or 1-3 carbon atoms. In specificembodiments, HG is a group as in FIG. 1-1, 1-2, 1-3, 2-1, 2-2, or 2-3and A is a group in FIG. 3. In specific embodiments, HG is a group as inFIG. 1-1, 1-2, 1-3, 2-1, 2-2, or 2-3 and A is a group in FIG. 3 and

W is

In specific embodiments, A is one of A1-A13 (FIG. 3-1). In specificembodiments, A is one of A1-A13 (FIG. 3-1) and n is 0. In specificembodiments, A is one of A1-A13 (FIG. 3-1), n is 0, q is 1 and Y is —CO—or —CO—CH₂—CO—. In specific embodiments, A is one of A1-A13 (FIG. 3-1),n is 0, q is 1, Y is —CO— or —CO—CH₂—CO—, and W is —NH—. In specificembodiments, A is one of A1-A13 (FIG. 3-1), n is 0, q is 1, Y is —CO— or—CO—CH₂—CO—, and W is

In specific embodiments, A is one of A1-A13 (FIG. 3), n is 0, q is 1, Yis —CO— or —CO—CH₂—CO—, W is —NH— and m is 0. In specific embodiments, Ais one of A1-A13 (FIG. 3), n is 0, q is 1, Y is —CO— or —CO—CH₂—CO—, Wis —NH— and m is 1.

In specific embodiments, A is a branched or straight chain alkyl oralkenyl group. In specific embodiments, A is a branched or straightchain alkyl or alkenyl and n is 0. In specific embodiments, A is abranched or straight chain alkyl or alkenyl, n is 0, q is 1 and Y is—CO— or —CO—CH₂—CO—. In specific embodiments, A is a branched orstraight chain alkyl or alkenyl, n is 0, q is 1, Y is —CO— or—CO—CH₂—CO— and W is —NH—. In specific embodiments, A is a branched orstraight chain alkyl or alkenyl, n is 0, q is 1, Y is —CO— or—CO—CH₂—CO—, and W is

In specific embodiments, A is a branched or straight chain alkyl oralkenyl, n is 0, q is 1, Y is —CO— or —CO—CH₂—CO—, W is —NH— and m is 0.In specific embodiments, A is a branched or straight chain alkyl oralkenyl n is 0, q is 1, Y is —CO— or —CO—CH₂—CO—, W is —NH— and m is 1.

In specific embodiments, HG is a group as in FIG. 1-1, 1-2, 2-1, 2-2, or2-3 and W is NH and m is 1. In specific embodiments, HG is a group as inFIG. 1-1, 1-2, 2-1, 2-2, or 2-3, L1 is —CH₂— or —CH₂—CH₂—, L2 is—(CH₂)0-1- and q is 1. In specific embodiments, HG is a group as in FIG.1-1, 1-2, 2-1, 2-2, or 2-3, L1 is —CH₂— or —CH₂—CH₂—, L2 is —(CH₂)₀₋₁—and q is 1 and Y is —CO— or —CO—CH₂—CO—. In specific embodiments, HG isa group as in FIG. 1-1, 1-2, 2-1, 2-2, or 2-3, and L1 is —CH₂— or—CH₂—CH₂—, L2 is —(CH₂)₀₋₁— and q is 1 and Y is —CO—.

In specific embodiments, HG is a group as in FIG. 1-1, 1-2, 2-1, 2-2, or2-3 and A is a branched or straight chain alkyl or alkenyl and W is —NH—and m is 1. In specific embodiments, HG is a group as in FIG. 1-1, 1-2,2-1, 2-2, or 2-3 and A is a branched or straight chain alkyl or alkenyl,L1 is —CH₂— or —CH₂—CH₂—, L2 is —(CH₂)₀₋₁— and q is 1. In specificembodiments, HG is a group as in FIG. 1-1, 1-2, 2-1, 2-2, or 2-3 and Ais a branched or straight chain alkyl or alkenyl, L1 is —CH₂— or—CH₂—CH₂—, L2 is —(CH₂)₀₋₁— and q is 1 and Y is —CO— or —CO—CH₂—CO—. Inspecific embodiments, HG is a group as in FIG. 1-1, 1-2, 2-1, 2-2, or2-3 and A is a branched or straight chain alkyl or alkenyl, and L1 is—CH₂— or —CH₂—CH₂-L2 is —(CH₂)₀₋₁— and q is 1 and Y is —CO—.

In specific embodiments, HG is a group as in FIG. 1-1, 1-2, 2-1, 2-2, or2-3 and A is a group in FIG. 3-1 and W is NH and m is 1. In specificembodiments, HG is a group as in FIG. 1-1, 1-2, 2-1, 2-2, or 2-3 and Ais a group in FIG. 3, L1 is —CH₂— or —CH₂—CH₂—, L2 is —(CH₂)₀₋₁— and qis 1. In specific embodiments, HG is a group as in FIG. 1-1, 1-2, 2-1,2-2, or 2-3 and A is a group in FIG. 3, L1 is —CH₂— or —CH₂—CH₂—, L2 is—(CH₂)₀₋₁— and q is 1 and Y is —CO— or —CO—CH₂—CO—. In specificembodiments, HG is a group as in FIG. 1-1, 1-2, 2-1, 2-2, or 2-3 and Ais a group in FIG. 3, with the exception that A is not the same group asHG and L1 is —CH₂— or —CH₂—CH₂—, L2 is —(CH₂)₀₋₁— and q is 1 and Y is—CO—.

In specific embodiments, HG is a group as in FIG. 1-1, 1-2, 1-3, 2-1,2-2, or 2-3 and A is a group in FIG. 3-1 with the exception that A isnot the same group as HG.

In specific embodiments, HG is P1-P50. In specific embodiments, HG isP1-P50 and L2 is —(CH₂)₀₋₂— and may be substituted on one carbon with analkyl group having 1-3 carbon atoms. In specific embodiments, HG isP1-P50; L2 is —(CH₂)₀₋₂— and is optionally substituted on one carbonwith an alkyl group having 1-3 carbon atoms, and m is 0. In specificembodiments, HG is P1-P50; L2 is —(CH₂)₀₋₂— and may be substituted onone carbon with an alkyl group having 1-3 carbon atoms, and m is 1. Inspecific embodiments, HG is P1-P50; L2 is —(CH₂)₀₋₂— and may besubstituted on one carbon with an alkyl group having 1-3 carbon atoms,and m is 1 and W is NH. In specific embodiments, HG is P1-P50; L2 is—(CH₂)₀₋₂— and may be substituted on one carbon with an alkyl grouphaving 1-3 carbon atoms, and m is 1, W is NH and q is 1. In specificembodiments, HG is P1-P50; L2 is —(CH₂)₀₋₂— and may be substituted onone carbon with an alkyl group having 1-3 carbon atoms, and m is 1, W is—NH—, q is 1 and Y is —CO— or —CO—CH₂—CO—. In specific embodiments HG isP1-P50 and L1 is —(CH2)0-2-. In specific embodiments HG is P1-P50 and nis 0.

HG groups may be unsubstituted. HG groups may be substituted. Optionalsubstitution on HG groups includes substitution with one or morenon-hydrogen substituents selected from the group consisting of halogen,nitro, hydroxyl, nitrile, azide, —R, —OR, —COOR, —OCOR, —COR, —OCOOR,—CON(R)₂, —OCON(R)₂, —N(R)₂, —SR, —SO₂R, —SOR, —SO₂N(R)₂, wherein each Ris independently selected from the group consisting of hydrogen, anamine group, a substituted or unsubstituted unbranched C1-C12 acyclicaliphatic group, a substituted or unsubstituted branched C1-C12 acyclicaliphatic group, a substituted or unsubstituted C3-C8 cycloalkyl group,a substituted or unsubstituted C3-C8 cycloalkenyl group, a fluorinatedC1-C12 alkyl group, a substituted or unsubstituted aryl group, asubstituted or unsubstituted heterocyclic group, and a protecting group,where two R groups on the same substituent optionally form a 4-8 memberring (carbon ring or a carbon ring with 1-3 heteroatom ring members);additionally, two X, X1 or X2 groups, particularly two such groups onadjacent ring positions can form a 4-8 member ring. Specificsubstituents include among others optionally substituted alkyl groupshaving 1-3 carbon atoms.

In specific embodiments, X, X1 or X2 represent one or more halogens,nitro, azide, nitrile, alkyl groups particularly those having 1-3 carbonatoms, —OR, —COOR, —SO₂—R, —SR, or —N(R)₂, particularly where R ishydrogen or an alkyl group having 1-3 carbon atoms.

In specific embodiments, one or more carbons of L1 can be substitutedwith an alkyl group having 1-3 carbon atoms, a hydroxyl or amine groupor a halogen, particularly a fluorine. In a more specific embodiment onecarbon of L1 can be substituted with one non-hydrogen substituent. In aspecific embodiment L1 is —CH(R′)— where R′ is an alkyl group. In aspecific embodiment L1 is —(CF₂)_(p1)—.

In specific embodiments, one or more carbons of L2 can be substitutedwith an alkyl group having 1-3 carbon atoms, a hydroxyl or amine groupor a halogen, particularly a fluorine. In a more specific embodiment onecarbon of L2 can be substituted with one non-hydrogen substituent. In aspecific embodiment L2 is —CH(R′)— where R′ is an alkyl group. In aspecific embodiment L2 is —(CF₂)_(p1)—.

The invention also provides a compound of formula II:

or a pharmaceutically acceptable salt or ester thereof

where variables are defined as for formula I. In an embodiment, q is 0.In an embodiment q is 1. In an embodiment, q is 1 and Y is —CO—,—CO—CH₂—CO— or —CO—CH₂—C(Y1)-.

In embodiments of formula II, A is a branched or unbranched C1-C12acyclic aliphatic group. More specifically A is a branched or unbranchedalkyl or alkenyl group having 1-15 carbon atoms. In more specificembodiments of formula II where A is a branched or unbranched C1-C12acyclic aliphatic group, n is 0, q is 0 and L1 is —(CH₂)_(p1)—, where p1is 0-6. In additional specific embodiments of formula II where A is abranched or unbranched C1-C12 acyclic aliphatic group, n is 0, q is 1, Yis —CO— or —CO—CH₂—C(Y1)-, L1 is —(CH₂)_(p1)—, where p1 is 0-6. In morespecific embodiments, A is a branched or unbranched alkyl group having1-12 carbon atoms.

In embodiments of formula II, A is an optionally substituted aryl group.In more specific embodiments, q is 0, L1 is —(CH₂)_(p)— where p is 0-6and A is an optionally substituted aryl group, particularly anoptionally substituted phenyl, biphenyl or naphthyl group. In additionalspecific embodiments, q is 0, L1 is —(CH₂)_(p)—, where p is 1-3, and Ais an optionally substituted aryl group, particularly an optionallysubstituted phenyl, biphenyl or naphthyl group. In additionalembodiments, the phenyl, biphenyl or naphthyl group is unsubstituted orsubstituted with one or more halide, nitro, hydroxyl, nitrile, azide,—OR, —N(R)₂, —SR, or —SO₂R groups, where R is an alkyl group having 1-3carbon atoms.

In embodiments of formula II, HG is an optionally substituted phenyl,naphthyl, cyclopentyl, cyclohexyl, cyclohexenyl, furyl, or group havingformula:

where variables are as defined above and in specific embodiments, r is 1or 2. In additional embodiments, the ring is unsubstituted and R′ ishydrogen. In additional embodiments, R′ is an alkyl group having 1-3carbon atoms. In additional embodiments, the ring carries 1-3substituents, particularly optionally substituted alkyl groups having1-3 carbon atoms. Preferred optional substitution for phenyl, naphthyl,cyclopentyl, cyclohexyl, or cyclohexenyl HG groups is one or morehalogen, nitro, or alkoxy (having 1-3 carbon atoms). In specificembodiments, HG is:

where r, X and R′ are as defined above. In specific embodiments, r is 1.IN specific embodiments, R′ is H. In specific embodiments, R′ isoptionally substituted C1-C3 alkyl. In specific embodiments, X is 1-3substituents on the ring. In specific embodiments, r is 1 and X is 1 or2 substituents on the ring. In specific embodiments, X is 1 or 2optionally substituted alkyl groups having 1-3 carbon atoms.

Compounds of this invention can be optically active, racemic,enantiomerically pure or mixtures of enantiomers. HG may have opticallyactive carbons and may exist as enantiomeric pairs. For example, HG offormula:

can be in the enantiomeric forms:

Note that carbons in the HG ring other than that shown may be opticallyactive dependent upon X substitution.

The invention also provides a compound of formula III:A-[Z]_(n)-L1-[Y]_(q)—NH—[V]_(m)-L2-HGor a pharmaceutically acceptable salt or ester thereof,

-   where variables are defined as for formula I. In specific    embodiments of formula III, m is 0. In other specific embodiments, n    is 0. In other specific embodiments, m and n are both 0. In specific    embodiments, m is 0 and q is 1. In more specific embodiments, m is    0, q is 1 and Y is —CO—, —CO—CH₂—CO— or —CO—CH₂—C(Y1)-. In more    specific embodiments, m is 0, q is 1 and Y is —CO—, —CO—CH₂—CO— or    —CO—CH₂—C(Y1)-, and A is an optionally substituted aryl group,    particularly an optionally substituted phenyl group. In more    specific embodiments, m is 0, q is 1 and Y is —CO—, —CO—CH₂—CO— or    —CO—CH₂—C(Y1)-, and A is an optionally substituted branched or    unbranched C₁-C₁₂ acyclic aliphatic group. In specific embodiments    HG is a group of any of FIG. 1-1, 1-2, 1-3, 2-1, 2-2 or 2-3. In    specific embodiments, HG is:

where r, X and R′ are as defined above. In specific embodiments, r is 1.In specific embodiments, R′ is H. In specific embodiments, R′ isoptionally substituted C1-C3 alkyl. In specific embodiments, X is 1-3substituents on the ring. In specific embodiments, r is 1 and X is 1 or2 substituents on the ring. In specific embodiments, X is 1 or 2optionally substituted alkyl groups having 1-3 carbon atoms.

The invention also provides a compound of formula IV:

or a pharmaceutically acceptable salt or ester thereof,

-   where variables are defined as for formula I. In specific    embodiments, R_(N) is hydrogen. In specific embodiments, q is 1 and    Y is —CO—, —CO—CH₂—CO— or —CO—CH₂—C(Y1)-. In specific embodiments,    HG is an optionally substituted aryl group, particularly an    optionally substituted phenyl group. In specific embodiments, A is A    is an optionally substituted aryl group, particularly an optionally    substituted phenyl group. In specific embodiments, A is an    optionally substituted branched or unbranched C₁-C₁₂ acyclic    aliphatic group, particularly an optionally substituted branched or    unbranched alkyl or alkenyl group having 1 to 12 carbon atoms. In    specific embodiments HG is a group of any of FIG. 1-1, 1-2, 2-1, 2-2    or 2-3. In specific embodiments, HG is:

where r, X and R′ are as defined above. In specific embodiments, r is 1.IN specific embodiments, R′ is H. In specific embodiments, R′ isoptionally substituted C1-C3 alkyl. In specific embodiments, X is 1-3substituents on the ring. In specific embodiments, r is 1 and X is 1 or2 substituents on the ring. In specific embodiments, X is 1 or 2optionally substituted alkyl groups having 1-3 carbon atoms.

The invention also provides a compound of formula V:

or a pharmaceutically acceptable salt or ester thereof, where variablesare defined as for formula I. In specific embodiments, R_(E) is anunsubstituted alkyl group having 1-6 carbon atoms. In specificembodiments, R_(E) is methyl or ethyl. In specific embodiments, A is abranched or straight-chain aliphatic group having 1-12 carbon atoms. Inspecific embodiments, A is a branched or straight-chain alkyl grouphaving 1-12 carbon atoms. In specific embodiments, A is an optionallysubstituted phenyl group. In specific embodiments, A is a phenyl groupsubstituted with one or more halogens, nitro groups, alkoxy groupshaving 1-3 carbon atoms, or one or more trifluoroethyl groups. Inspecific embodiments W is —NH—. In specific embodiments L1 and L2 areindependently either —CH₂— or —CH₂—CH₂—. In a specific embodiment L2 is—CH(CH₃)—. In specific embodiments, Y is —CO—, —CO—CH₂—CO—, —NH—CO—,—CO—CH₂—C(Y1)-. In specific embodiments, Y is —CO—, or —CO—CH₂—CO. Inspecific embodiments, q is 1. In specific embodiments, n is 0. Inspecific embodiments, m is 0. In specific embodiments, n and m are 0 andq is 1. In specific embodiments Y is —CO— or —CO—CH₂—CO—.

The invention also provides a compound of formula VI:

where R_(F) is an optionally substituted a branched or unbranched C₁-C₁₂acyclic aliphatic group, L2, V and m are as defined above, f is 0 or 1to show the absence of presence of the CO group, and HG is a head groupas defined in formula I. In specific embodiments, m is 0. In specificembodiments m is 1. In specific embodiments L2 is —CH₂— or —CH₂—CH₂—. Inspecific embodiments, HG can be any group as in FIG. 1-1, 1-2 or 1-3. Inother specific embodiments, HG is an optionally substituted phenylgroup. In specific embodiments, L2 is —CH₂— and HG is an optionallysubstituted phenyl group. In specific embodiments, m is 1, L2 is —CH₂—and HG is an optionally substituted phenyl group. In specificembodiments, m is 0, L2 is —CH₂— and HG is an optionally substitutedphenyl group. In specific embodiments R_(F) is a branched orstraight-chain alkyl group. In specific embodiments R_(E) is a branchedor straight-chain alkenyl group having one or two double bonds. Inspecific embodiments, f is 1 and m is 0. In specific embodiments, f is 0and m is 0. In specific embodiments, f and m are both 1. In specificembodiments, f is 0 and m is 1. In specific embodiments, HG is a phenylgroup substituted with 1 to 5 halogens, particularly bromine, chlorineor fluorine. In specific embodiments, HG is a phenyl group substitutedwith 1 to 5 fluorines. In specific embodiments, HG is a phenyl groupsubstituted with 1 or 2 alkoxy groups having 1-3 carbon atoms. Inspecific embodiments, HG is a phenyl group substituted with 1-3 nitrogroups. In specific embodiments, HG is a furyl group, particularly a1-furyl group. In specific embodiments, m is 1, f is 1, L2 is —CH₂— or—CH₂—CH₂— and HG is selected from HG groups of FIG. 1-1, 1-2, 1-3, 2-1,2-2, 2-3 or 2-4. In specific embodiments, m is 1, f is 0, L2 is —CH₂— or—CH₂—CH₂— and HG is selected from HG groups of 1-1, 1-2, 1-3, 2-1, 2-2,2-3 or 2-4. In specific embodiments, m is 0, L2 is —CH₂— or —CH₂—CH₂—and HG is selected from HG groups of FIG. 1-1, 1-2, 1-3, 2-1, 2-2 or2-3. In specific embodiments, m is 0, f is 1, L2 is —CH₂— or —CH₂—CH₂—and HG is selected from HG groups of FIG. 11-1, 1-2, 1-3, 2-1, 2-2, 2-3or 2-4. In specific embodiments, m is 0, f is 0, L2 is —CH₂— or—CH₂—CH₂— and HG is selected from HG groups of FIG. 1-1, 1-2, 1-3, 2-1,2-2, 2-3 or 2-4. In specific embodiments, HG is:

where r, X and R′ are as defined above. In specific embodiments, r is 1.In specific embodiments, R′ is H. In specific embodiments, R′ isoptionally substituted C1-C3 alkyl. In specific embodiments, X is 1-3substituents on the ring. In specific embodiments, r is 1 and X is 1 or2 substituents on the ring. In specific embodiments, X is 1 or 2optionally substituted alkyl groups having 1-3 carbon atoms.

The present invention provides compounds and methods for modulation ofquorum sensing of bacteria. In an embodiment, the compounds of thepresent invention are able to act as replacements for naturallyoccurring bacterial quorum sensing ligands in a ligand-protein bindingsystem; that is, they imitate the effect of natural ligands and producean agonistic effect. In another embodiment, the compounds of the presentinvention are able to act in a manner which disturbs or inhibits thenaturally occurring ligand-protein binding system in quorum sensingbacteria; that is, they produce an antagonistic effect. The presentinvention also provides methods of increasing or reducing the virulenceof quorum sensing bacteria. In one aspect, the method comprisescontacting a bacterium with an effective amount of a compound of thepresent invention. In another aspect, the method comprises contacting abacterium with a therapeutically effective amount of a pharmaceuticallyacceptable salt or ester of the compounds of the present invention. Inyet another aspect, the method comprises contacting a bacterium with aprecursor which can form an effective amount of a compound of thepresent invention.

The present invention provides compositions for modulation of quorumsensing of bacteria which comprises one or more compounds of thisinvention, particularly one or more compounds of formulas I to VIherein. The compositions herein can further comprise an appropriatecarrier, particularly a pharmaceutically acceptable carrier fortherapeutic applications. In applications herein, one or more compoundsof the invention can be compounds with one or more antibacterialcompounds.

In an embodiment, the methods of the present invention can be used fordisrupting a biofilm formed by a quorum sensing bacterium. A method ofthe present invention for disrupting a biofilm comprises contacting thebiofilm with an effective amount of a compound of the present invention.In an embodiment, the methods of the present invention can be used todiminish or inhibit biofilm production. Alternatively, the methods ofthe present invention can be used for causing a quorum sensing bacteriumto initiate or enhance biofilm production. Initiation or enhancement ofbiofilm formation of beneficial bacteria (those, for example, thatprovide a health benefit or are used in production of a valuableproduct) can facilitate or enhance such a health benefit or can be usedto enhance or improve production of desirable valuable products. In aspecific embodiment, compounds which activate quorum sensing ofbeneficial gut bacterial can provide a probiotic effect.

In an embodiment, the methods of the present invention can be used forinhibiting or diminishing the symbiotic behavior of a quorum sensingbacteria. In another embodiment, the methods of the present inventioncan be used for stimulating, initiating, or enhancing the symbioticbehavior of a quorum sensing bacteria.

In another embodiment of the methods, the compounds of the presentinvention can be administered to a subject to initiate modulation ofquorum sensing of bacteria. In an embodiment, the administration of aneffective amount of a compound of the present invention to a subject caninitiate or enhance the symbiotic behavior of quorum sensing bacteria inthe subject. In an embodiment, the administration of an effective amountof a compound of the present invention to a subject can disrupt abiofilm of quorum sensing bacteria in the subject. In an embodiment, theadministration of an effective amount of a compound of the presentinvention to a subject can initiate or enhance the symbiotic behavior ofa target species or a selected strain of a target species of quorumsensing bacteria in the subject. In an embodiment, the administration ofan effective amount of a compound of the present invention to a subjectcan regulate the virulence of quorum sensing bacteria in the subject. Inan embodiment, the administration of an effective amount of a compoundof the present invention to a subject can regulate the virulence of atarget species or a selected strain of a target species of quorumsensing bacteria in the subject.

The methods of the present invention also provide for regulation of thelevel of virulence of quorum sensing bacteria. In an embodiment, one ormore compounds of the present invention is brought into contact with aquorum sensing bacteria to selectively regulate the virulence of thebacteria. In an embodiment, a mixture of the compounds of the presentinvention is brought into contact with a quorum sensing bacteria toselectively regulate the virulence of the bacteria. The amount of eachcompound in the mixture is that amount effective to achieve a desiredeffect on regulation of virulence. The methods of the present inventionalso provide for regulation of the production of a biofilm by quorumsensing bacteria. In an embodiment, one or more compounds of the presentinvention is brought into contact with a quorum sensing bacteria orbacterial biofilm to selectively regulate the biofilm production by thebacteria. In an embodiment, a mixture of the compounds of the presentinvention is brought into contact with a quorum sensing bacteria orbacterial biofilm to selectively regulate the biofilm production by thebacteria. The amount of each compound in the mixture is that amounteffective for desired regulation of biofilm formation.

The methods of the present invention also provide for regulation of thevirulence, biofilm production, or symbiotic behavior of a quorum sensingbacteria by contacting the bacteria with a photoactive compound andilluminating the bacteria and photoactive compound. In an embodiment,illuminating a photoactive compound of the present invention can changethe agonistic or antagonistic behavior of the compound.

In an embodiment, the present invention provides a surface coating orpolymer having incorporated therein a compound of the present invention.The amount of compound or polymer in the surface coating is thatsufficient to provide antimicrobial or antifouling effect. In anembodiment, the compounds of the present invention are useful as anantimicrobial and/or antifouling agent. Compounds of the presentinvention are further useful in a medical, scientific, and/or biologicalapplication. In one aspect, the present invention provides a compositioncomprising one or more compounds of the present invention and a carrieror diluent. In a preferred embodiment, the carrier or diluent comprisesa liquid. Such a liquid may comprises an aqueous solvent or anon-aqueous solvent. An exemplary solvent comprises one or more organicsolvents. The carrier or diluent may also comprise an ionic liquid. Inan embodiment of this aspect, the composition comprises an organic orinorganic polymeric substance. The polymeric substance may comprise oneor more compounds of the present invention, admixed with a polymer,bound to a polymer, or adsorbed on to a polymer. In an exemplaryembodiment of this aspect, the composition is in the form of a solutionor suspension of said at least one compounds of the present invention,preferably in an aerosol or powder formulation.

In an embodiment of this aspect, the composition is formulated as adisinfectant or cleaning formulation. In another embodiment, thecomposition is in the form of a powder, a solution, a suspension, adispersion, an emulsion, or a gel. In an exemplary embodiment, thecomposition is in the form of a pharmaceutical composition comprising apharmaceutically acceptable carrier, diluent, and/or excipient and oneor more compounds of the present invention. The composition may be in aform suitable for parenteral or non-parenteral administration. Apreferred composition may be formulated for topical, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, ophthalmic, or oral administration. In an embodiment of thisaspect the composition is formulated for administration by infusion orbolus injection, absorption through epithelial or mucocutanous liningsand may be administered together with other biologically active agents.In an embodiment, the composition may further be formulated for use inan inhaler or nebulizer.

In another aspect, the present invention provides a method of treatingan infection in a human or animal subject, the method comprisingadministration to the subject of an effective amount of one or morecompounds of the present invention. In an embodiment, the treatment istherapeutic or prophylactic. In an embodiment, the method furthercomprises administering one or more pharmaceutically acceptableantibacterial compounds to the subject, prior to, at the same time as orafter administration of the one or more compounds of this invention.

In a related embodiment, the present invention provides a method oftreating an infection or condition in a subject that is characterized bybiofilm formation, the method comprising administering one or morecompounds of the present invention. In an embodiment, the method furthercomprises administering one or more pharmaceutically acceptableantibacterial compounds to the subject, prior to, at the same time as orafter administration of the one or more compounds of this invention. Inan embodiment, the condition is cystic fibrosis. In an embodiment, thecondition is dental caries, periodonitis, otitis media, muscularskeletal infections, necrotizing fasciitis, biliary tract infection,osteomyelitis, bacterial prostatitis, native valve endocarditis, cysticfibrosis pneumonia, or meloidosis. In an embodiment, the condition is anosocomial infection; preferably the infection is ICU pneumonia or aninfection associated with sutures, exit sites, arteriovenous sites,scleral buckles, contact lenses, urinary catheter cystitis, peritonealdialysis (CAPD) peritonitis, IUDs, endotracheal tubes, Hickmancatheters, central venous catheters, mechanical heart valves, vasculargrafts, biliary stent blockage, orthopedic devices, or penileprostheses. In an embodiment, the infection is a skin infection, a burninfection, or a wound infection. According to this aspect, the subjectmay preferably be an immunocompromised individual.

The present invention further provides a method for treating orpreventing biofilm formation on a surface, the method comprisingcontacting said surface with one or more compounds in an amounteffective for affecting biofilm formation of the present invention. Inan embodiment, the method further comprises contacting the surface withone or more antibacterial compounds appropriate for the application,prior to, at the same time as or after contact with the one or morecompounds of this invention. In an embodiment, the surface is anon-biological surface. In an embodiment, the surface is a naturalsurface. In an embodiment, the surface is a surface of a plant, seed,wood, fiber or hair. In an embodiment, the surface is a biologicalsurface; preferably the surface is a surface of a tissue, membrane, orskin. In an embodiment, the surface is a hard surface; preferably thesurface comprises a metal, an organic polymer, an inorganic polymer, anatural elastomer, a synthetic elastomer, glass, wood, paper, concrete,rock, marble, gypsum, or ceramic. In an embodiment, the said surface iscoated or wherein the surface is a coating; in a preferred embodiment,the coating comprises enamel, varnish, or paint.

In an embodiment of this aspect, the surface is a soft surface, and maybe the surface of a fiber comprising a yarn, a textile, a vegetablefiber, or rock wool. In another embodiment, the surface is a poroussurface. In an embodiment, the surface is a surface of process equipmentor components of cooling equipment. In a preferred embodiment, theprocess equipment is or is a component of a cooling tower, a watertreatment plant, a dairy processing plant, a food processing plant, achemical process plant, or a pharmaceutical process plant. In apreferred embodiment the surface is that of a filter or a membranefilter.

In an embodiment of this aspect, the surface is a surface of a toiletbowl, a bathtub, a drain, a high-chair, a counter top, a vegetable, ameat processing room, a butcher shop, food preparation areas, an airduct, an air-conditioner, a carpet, paper or woven product treatment, adiaper, personal hygiene products and a washing machine. In anotherembodiment, the surface is an industrial surface or a medical surface;preferably the surface is a surface in a hospital, a veterinaryhospital, a mortuary, or a funeral parlor.

In another aspect, the compounds of the present invention are useful asa component of a dentifrice, a mouthwash, or a composition for thetreatment of dental caries; for treatment of acne; or for cleaningand/or disinfecting contact lenses. The compounds of the presentinvention are further useful for incorporation into the surface of amedical device or an implant device. Preferably the implant device is anartificial heart valve, hip joint, an indwelling catheter, pacemaker, orsurgical pin. The compounds of the present invention are further usefulas an antifouling coating. The present invention further provides anoptical lens, wherein at least a part of a surface of the lens isassociated with one or more compounds of the present invention.Preferably, the optical lens is a contact lens.

In another aspect, the present invention provides a biofilm removing orinhibiting composition comprising one or more compounds of the presentinvention in an amount effective for removing or inhibiting biofilmformation and a vehicle or carrier, wherein the amount of the mixture iseffective to remove or disrupt a bacterial biofilm or inhibit normalbiofilm formation. An embodiment of this aspect may further comprise asurfactant selected from the group consisting of an anionic surfactant,a nonionic surfactant, an amphoteric surfactant, a biologicalsurfactant, and any combination of these; or a compound selected fromthe group consisting of an antibacterial which includes among others abiocide, a fungicide, an antibiotic, and any combination of these.

In another aspect, the present invention provides a method of removing abiofilm from a surface, the method comprising the step of administeringa cleaning-effective amount of one or more compounds of the presentinvention to a biofilm-containing surface. A preferred method of thisaspect comprises the step of administering an effective amount of one ormore compounds of the present invention to the surface, wherein theamount is effective to prevent biofilm formation. Such a surface may bea hard or rigid surface or a surface selected from the group consistingof glazed ceramic, porcelain, glass, metal, wood, chrome, plastic,vinyl, composite materials (such as Formica® (Formica Corporation,Cincinnati, Ohio), and the surface of a drainpipe. In an embodiment, thesurface is a soft or flexible surface, or the surface is selected fromthe group consisting of a shower curtain or liner, upholstery, laundry,clothing, and carpeting. In an embodiment, the surface is a biologicalsurface and the effective amount is a therapeutically effective amountfor application to the biological surface for inhibiting biofilmformation. The compounds of the present invention are useful inparticular, for removing or disrupting a biofilm produced by a bacteriumof the class Pseudomonas, a bacterium is of the species Pseudomonasaeruginosa, or an organism selected from the group consisting ofbacteria, algae, fungi and protozoa. In a specific aspect, this methodfurther comprises a step of applying or administering to abiofilm-containing surface an antibacterial compound before, at the sametime as or after applying or administering the one or more compounds ofthis invention.

In another aspect, the invention provides a medicament for treating aninfection or for disruption of a biofilm which comprises one or more ofthe compounds of this invention e.g., those of formulas I-VI, and amethod for making a medicament which comprises one or more of thecompounds of this invention. In particular, the method comprises thestep of combining one or more compounds of this invention with apharmaceutically acceptable carrier to form a pharmaceutical compositionfor treatment of infection and/or biofilm formation. In anotherparticular embodiment, the method further comprises combining anantibacterial compound appropriate for the application to a medicamentalong with one or more compounds of this invention.

Without wishing to be bound by any particular theory, there can bediscussion herein of beliefs or understandings of underlying principlesrelating to the invention. It is recognized that regardless of theultimate correctness of any mechanistic explanation or hypothesis, anembodiment of the invention can nonetheless be operative and useful.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 (2 pages) illustrates exemplary HG groups.

FIG. 2 (3 pages) illustrates exemplary HG groups.

FIG. 3 illustrates exemplary A groups.

FIG. 4 provides structures (and reference numbers) of exemplarynon-homoserine lactone based autoinducer analogs synthesized by themethod highlighted in Scheme 1.

FIG. 5A is a bar graph showing the results of agonism assay for initialheterocyclic and carbocyclic library (Scheme 1) shown as a percent ofthe positive control. The black bars are DH5α (pJN105L+pSC11) and thegrey bars are PA01 MW1 (pUM15). Agonism positive control=activity of thereporter strain at full turn on for the strain. Full turn on for eachstrain: DH5α (pJN105L+pSC11)—100 nM OdDHL; PA01 MW1 (pUM15)—100 μMOdDHL. Negative control (Neg)=bacteria in the absence of natural andsynthetic ligand. Error bars=standard deviation of the mean oftriplicate samples.

FIG. 5B is a bar graph showing the results of agonism assay for initialheterocyclic and carbocyclic library (Scheme 1) shown as a percent ofthe positive control. Antagonism positive control (Pos)=activity of thereporter strain in the absence of synthetic ligand at the EC50 value forthe strain. Strain EC50 values: DH5α (pJN105L+pSC11)—10 nM OdDHL; PA01MW1 (pUM15)—1 μM OdDHL. Negative control (Neg)=bacteria in the absenceof natural and synthetic ligand. Error bars=standard deviation of themean of triplicate samples.

FIG. 6 provides structures (with reference numbers) of the racemicthiolactone library prepared as illustrated in Scheme 2.

FIGS. 7A and 7B are bar grafts presenting results of agonism (7A) andantagonism (7B) assays for the racemic thiolactone library (FIG. 6). Thebiological testing conditions were the same as described in FIGS. 5A and5B, respectively.

FIG. 8 provides structures (with reference numbers) of the enantiopurethiolactone library and EDC couplings.

FIGS. 9A-9H are bar grafts comparing agonism and antagonism of theracemic and enantiopure compounds of Libraries of FIG. 6 and FIG. 8. Allsynthetic ligands were tested at 10 μM using standard methods describedin FIGS. 5A and 5B.

FIGS. 10A and B are graphs comparing the functional half-lives ofautoinducers as described in Example 4.

FIG. 11 provides structures (with reference numbers) of compounds havingglycine ethyl ester structures.

FIGS. 12A and 12B are bar graphs with results of activity assays of theglycine ethyl ester library (for agonism 12A and antagonism 12 B)according to the assay conditions described in FIGS. 5A and 5B.

FIG. 13 provides structures (with reference numbers) of an exemplarylibrary having cyclopentyl amine head groups.

FIG. 14 provides structures (with reference numbers) of an exemplarylibrary having aniline head groups.

FIGS. 15A and 15B are bar graphs with results of activity assays of thecompounds of FIGS. 13 and 14 (for agonism 15A and antagonism 15 B)according to the assay conditions described in FIGS. 5A and 5B.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the broadest meanings as commonly understood by one of ordinaryskill in the art to which this invention pertains. In addition,hereinafter, the following definitions apply:

Quorum sensing assays conducted as described herein can be used toassess whether or no a given compound of the invention is a quorumsensing agonist or antagonist of a given bacterium, particularly a givenGram-Negative bacterium. It will be appreciated by one of ordinary skillin the art that assays other than those described herein can be employedto assess activation of or inhibition of biofilm formation as well asthe effect of compounds of this invention on bacterial growth.

As defined herein, “contacting” means that a compound of the presentinvention is provided such that it is capable of making physical contactwith another element, such as a microorganism, a microbial culture, abiofilm, or a substrate. In another embodiment, the term “contacting”means that a compound of the present invention is introduced into asubject receiving treatment, and the compound is allowed to come incontact in vivo.

Compounds of this invention that disrupt bacterial quorum sensing andbiofilm formation can be used in combination with antimicrobial andantibacterial compounds (other than compounds which inhibit quorumsensing). The terms antimicrobial and antibacterial are employed broadlyherein to refer to any compound that exhibits a growing inhibitionactivity on a microorganism or bacterium, respectively. A subset of suchantimicrobial and antibacterial compounds are pharmaceuticallyacceptable for use in the treatment of humans and animals. A subset ofantimicrobial and antibacterial compounds are biocides. A subset ofantimicrobial and antibacterial compounds are antibiotics. In specificembodiments, compounds of the invention which are inhibitors or quorumsensing and biofilm formation are used to augment or facilitate theaction of convention antibiotic treatment. The invention providesmethods in which contact with or treatment with one or more quorumsensing compounds of the invention which inhibit quorum sensing iscombined with contract with or treatment with one or more antimicrobialor antibacterial compounds. The invention provides methods in whichcontact with or treatment with one or more quorum sensing compounds ofthe invention which inhibit quorum sensing is combined with contractwith or treatment with one or more antibiotics. Antibiotics includeamong others beta-lactam antibiotics, cephaosporins, clavulanic acid andderivatives thereof, aminoglycosides, tetracyclines, macrolideantibiotics.

Quorum sensing inhibitors of the invention can also generally becombined with antimicrobial agents, including antifungal agents, andantiviral agents.

In some cases, combination of one or more quorum sensing inhibitor ofthis invention with one or more antibacterial compound, antimicrobialcompound or antiviral agent can enhance the activity of one or moreantibacterial compound, antimicrobial compound or antiviral agent. Insome case the combination of one or more quorum sensing inhibitor withone or more antibacterial compound, antimicrobial compound or antiviralagent synergizes the activity of the one or more antibacterial compound,antimicrobial compound or antiviral agent.

One or more quorum sensing inhibitor compounds of this invention can becombined with one or more antibacterial compounds, one or moreantimicrobial compounds, one or more antiviral compounds and morespecifically one or more antibiotics in pharmaceutically acceptablecompositions useful for treatment of infections. Such pharmaceuticalcompositions typically further comprise a pharmaceutically acceptablecarrier. Such combination compositions and medicaments can be employedfor treatment of infection.

Contact with or treatment employing one or more quorum sensing inhibitorcompounds of this invention can be combined with contact with ortreatment with one or more antibacterial compounds, one or moreantimicrobial compounds, one or more antiviral compounds and morespecifically one or more antibiotics. In this case, contact or treatmentis with one or more separate pharmaceutical composition which may be putin contact with the area to be treated (e.g., applied to a surface,including a biological surface) or administered to a subject at the sametime or at different times. The quorum sensing inhibitor can be appliedor administered before, after or at the same time as the antibacterialcompound, antimicrobial compound or antiviral compound is applied oradministered.

Aliphatic groups include straight chain, branched, and cyclic groupshaving a carbon backbone having from 1 to 30 carbon atoms. Aliphaticgroups include alkyl groups, alkenyl groups, alkynyl groups, and arylgroups. Aliphatic groups are optionally substituted with one or morenon-hydrogen substituents. Substituted aliphatic groups include amongothers those which are substituted with aryl groups, which in turn canbe optionally substituted. Substituted aliphatic groups include fullyhalogenated or semihalogenated aliphatic groups, such as aliphaticgroups having one or more hydrogens replaced with one or more fluorineatoms, chlorine atoms, bromine atoms and/or iodine atoms. Substitutedaliphatic groups include fully fluorinated or semifluorinated aliphaticgroups, such as aliphatic groups having one or more hydrogens replacedwith one or more fluorine atoms. Aliphatic groups are optionallysubstituted with one or more protecting groups.

Alkyl groups include straight-chain, branched and cyclic alkyl groups.Alkyl groups include those having from 1 to 30 carbon atoms. Alkylgroups include small alkyl groups having 1 to 3 carbon atoms. Alkylgroups include medium length alkyl groups having from 4-10 carbon atoms.Alkyl groups include long alkyl groups having more than 10 carbon atoms,particularly those having 10-30 carbon atoms. Cyclic alkyl groupsinclude those having one or more rings. Cyclic alkyl groups includethose having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring andparticularly those having a 3-, 4-, 5-, 6-, 7-, or 8-member ring. Thecarbon rings in cyclic alkyl groups can also carry aliphatic groups.Cyclic alkyl groups can include bicyclic and tricyclic alkyl groups.Alkyl groups are optionally substituted with one or more non-hydrogensubstituents. Substituted alkyl groups include among others those whichare substituted with aliphatic groups, which in turn can be optionallysubstituted. Specific alkyl groups include methyl, ethyl, n-propyl,iso-propyl, cyclopropyl, n-butyl, s-butyl, t-butyl, cyclobutyl,n-pentyl, branched-pentyl, cyclopentyl, n-hexyl, branched hexyl, andcyclohexyl groups, all of which are optionally substituted. Substitutedalkyl groups include fully halogenated or semihalogenated alkyl groups,such as alkyl groups having one or more hydrogens replaced with one ormore fluorine atoms, chlorine atoms, bromine atoms and/or iodine atoms.Substituted alkyl groups include fully fluorinated or semifluorinatedalkyl groups, such as alkyl groups having one or more hydrogens replacedwith one or more fluorine atoms.

An alkoxy group is an alkyl group, as broadly discussed above, linked tooxygen and can be represented by the formula R—O—.

Alkenyl groups include straight-chain, branched and cyclic alkenylgroups. Alkenyl groups include those having 1, 2 or more double bondsand those in which two or more of the double bonds are conjugated doublebonds. Alkenyl groups include those having from 2 to 20 carbon atoms.Alkenyl groups include small alkenyl groups having 2 to 3 carbon atoms.Alkenyl groups include medium length alkenyl groups having from 4-10carbon atoms. Alkenyl groups include long alkenyl groups having morethan 10 carbon atoms, particularly those having 10-20 carbon atoms.Cyclic alkenyl groups include those having one or more rings. Cyclicalkenyl groups include those in which a double bond is in the ring or inan alkenyl group attached to a ring. Cyclic alkenyl groups include thosehaving a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring andparticularly those having a 3-, 4-, 5-, 6- or 7-member ring. The carbonrings in cyclic alkenyl groups can also carry aliphatic groups. Cyclicalkenyl groups can include bicyclic and tricyclic aliphatic groups.Alkenyl groups are optionally substituted with one or more non-hydrogensubstituents. Substituted alkenyl groups include among others thosewhich are substituted with aliphatic groups, which groups in turn can beoptionally substituted. Specific alkenyl groups include ethenyl,prop-1-enyl, prop-2-enyl, cycloprop-1-enyl, but-1-enyl, but-2-enyl,cyclobut-1-enyl, cyclobut-2-enyl, pent-1-enyl, pent-2-enyl, branchedpentenyl, cyclopent-1-enyl, hex-1-enyl, branched hexenyl, cyclohexenyl,all of which are optionally substituted. Substituted alkenyl groupsinclude fully halogenated or semihalogenated alkenyl groups, such asalkenyl groups having one or more hydrogens replaced with one or morefluorine atoms, chlorine atoms, bromine atoms and/or iodine atoms.Substituted alkenyl groups include fully fluorinated or semifluorinatedalkenyl groups, such as alkenyl groups having one or more hydrogensreplaced with one or more fluorine atoms.

Alkynyl groups include straight-chain, branched and cyclic alkynylgroups. Alkynyl groups include those having 1, 2 or more triple bondsand those in which two or more of the triple bonds are conjugated triplebonds. Alkynyl groups include those having from 2 to 20 carbon atoms.Alkynyl groups include small alkynyl groups having 2 to 3 carbon atoms.Alkynyl groups include medium length alkynyl groups having from 4-10carbon atoms. Alkynyl groups include long alkynyl groups having morethan 10 carbon atoms, particularly those having 10-20 carbon atoms.Cyclic alkynyl groups include those having one or more rings. Cyclicalkynyl groups include those in which a triple bond is in the ring or inan alkynyl group attached to a ring. Cyclic alkynyl groups include thosehaving a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring andparticularly those having a 3-, 4-, 5-, 6- or 7-member ring. The carbonrings in cyclic alkynyl groups can also carry aliphatic groups. Cyclicalkynyl groups can include bicyclic and tricyclic aliphatic groups.Alkynyl groups are optionally substituted with one or more non-hydrogensubstituents. Substituted alkynyl groups include among others thosewhich are substituted with alkyl or aryl groups, which groups in turncan be optionally substituted. Alkynyl groups include acetyl,methylacetyl, 1-pentynyl, and 2-pentynyl, all of which are optionallysubstituted. Substituted alkynyl groups include fully halogenated orsemihalogenated alkynyl groups, such as alkynyl groups having one ormore hydrogens replaced with one or more fluorine atoms, chlorine atoms,bromine atoms and/or iodine atoms. Substituted alkynyl groups includefully fluorinated or semifluorinated alkynyl groups, such as alkynylgroups having one or more hydrogens replaced with one or more fluorineatoms.

Aryl groups include groups having one or more 5- or 6-member aromatic orheteroaromatic rings. Aryl groups can contain one or more fused aromaticrings. Heteroaromatic rings can include one or more N, O, or S atoms inthe ring. Heteroaromatic rings can include those with one, two or threeN, those with one or two O, and those with one or two S, or combinationsof one or two or three N, O or S. Aryl groups are optionally substitutedwith one or more non-hydrogen substituents. Substituted aryl groupsinclude among others those which are substituted with alkyl or alkenylgroups, which groups in turn can be optionally substituted. Specificaryl groups include phenyl groups, biphenyl groups, pyridinyl groups,and naphthyl groups, all of which are optionally substituted.Substituted aryl groups include fully halogenated or semihalogenatedaryl groups, such as aryl groups having one or more hydrogens replacedwith one or more fluorine atoms, chlorine atoms, bromine atoms and/oriodine atoms. Substituted aryl groups include fully fluorinated orsemifluorinated aryl groups, such as aryl groups having one or morehydrogens replaced with one or more fluorine atoms. The term heteroarylis used for aryl groups having one or more heteroaromatic rings. Arylgroups include those that are not heteroaryl groups.

Arylalkyl groups are alkyl groups substituted with one or more arylgroups wherein the alkyl groups optionally carry additional substituentsand the aryl groups are optionally substituted. Specific alkylarylgroups are phenyl-substituted alkyl groups, e.g., phenylmethyl groups.Alkylaryl groups are alternatively described as aryl groups substitutedwith one or more alkyl groups wherein the alkyl groups optionally carryadditional substituents and the aryl groups are optionally substituted.Specific alkylaryl groups are alkyl-substituted phenyl groups such asmethylphenyl. Substituted arylalkyl groups include fully halogenated orsemihalogenated arylalkyl groups, such as arylalkyl groups having one ormore alkyl and/or aryl having one or more hydrogens replaced with one ormore fluorine atoms, chlorine atoms, bromine atoms and/or iodine atoms.

The term “heterocyclic or heterocyclyl” generically refers to amonoradical that contains at least one ring of atoms, which may be asaturated, unsaturated wherein one or more carbons of the ring arereplaced with a heteroatom (a non-carbon atom) To satisfy valence theheteroatom may be bonded to H or a substituent groups. Ring carbons maybe replaced with —O—, —S—, —NR—, —N═ among others. More specificallyheterocyclic groups can contain one or two 4-6 member rings wherein tworings may be fused. In specific embodiments, one or two rings of theheterocyclic group can contain one, two or three heteroatoms,particularly —O—, —S—, —NR— or —N═ and combinations of such heteroatoms.

Protecting groups are groups substituted onto an aliphatic hydrocarbonfor protection of one or more substituents, for example protection ofalcohols, amines, carbonyls, and/or carboxylic acids. Protecting groupsinclude, but are not limited to, acetyl groups, MEM groups, MOM groups,PMB groups, Piv groups, THP groups, TMS groups, TBDMS groups, TIPSgroups, methyl ethers, Cbz groups, BOC groups, FMOC groups, benzylgroups, PMP groups, acetal groups, ketal groups, acylal groups, dithianegroups, methyl esters, benzyl esters, t-butyl esters, and silyl esters.These and other protecting groups known in the art of organic synthesismay be optionally used as a substituent of an aliphatic group.

Optional substitution of aliphatic groups includes substitution with oneor more aliphatic groups, wherein the aliphatic groups are optionallysubstituted.

Optional substituents for aliphatic groups include among others: —R,—COOR, —COR, —CON(R)₂, —OCON(R)₂, —N(R)₂, —SR, —SO₂R, —SOR, —OCOOR,—SO₂N(R)₂, and —OR; wherein R is selected from the group consisting of,a hydrogen, a halogen, an amine group, a substituted or unsubstitutedunbranched C1-C12 acyclic aliphatic group, a substituted orunsubstituted branched C1-C12 acyclic aliphatic group, a substituted orunsubstituted C3-C8 cycloalkyl group, a substituted or unsubstitutedC3-C8 cycloalkenyl group, a fluorinated C1-C12 alkyl group, asubstituted or unsubstituted aryl group, a substituted or unsubstitutedheterocycle, a substituted or unsubstituted C1-C12 alkoxy group, afluorinated C1-C12 alkoxy group, a hydroxyl group, a nitrile group, anazide group, a nitro group, an acyl group, a thiol group, a protectinggroup, —COOR, —COR, —CON(R)₂, —OCON(R), —N(R)₂, —SR, —SO₂R, —SOR,—OCOOR, —SO₂N(R)₂, and —OR; additionally, R and R can form a ring.

Specific substituted alkyl groups include haloalkyl groups, particularlytrihalomethyl groups and specifically trifluoromethyl groups. Specificsubstituted aryl groups include mono-, di-, tri, tetra- andpentahalo-substituted phenyl groups; mono-, di-, tri-, tetra-, penta-,hexa-, and hepta-halo-substituted naphthalene groups; 3- or4-halo-substituted phenyl groups, 3- or 4-alkyl-substituted phenylgroups, 3- or 4-alkoxy-substituted phenyl groups, 3- or4-RCO-substituted phenyl, 5- or 6-halo-substituted naphthalene groups.More specifically, substituted aryl groups include acetylphenyl groups,particularly 4-acetylphenyl groups; fluorophenyl groups, particularly3-fluorophenyl and 4-fluorophenyl groups; chlorophenyl groups,particularly 3-chlorophenyl and 4-chlorophenyl groups; methylphenylgroups, particularly 4-methylphenyl groups, and methoxyphenyl groups,particularly 4-methoxyphenyl groups.

As to any of the above groups which contain one or more substituents, itis understood, that such groups do not contain any substitution orsubstitution patterns which are sterically impractical and/orsynthetically non-feasible. In addition, the compounds of this inventioninclude all stereochemical isomers arising from the substitution ofthese compounds.

The term “effective amount” is used generically herein to refer to theamount of a given compound or in case of a mixture the combined amountof mixture components that provides a measureable effect for a listedfunction. For example, in certain aspects of the invention, a compoundof the invention is contacted with an element in order to disrupt abiofilm and in this case, the effective amount or combined effectiveamount of the compound or compounds is that amount that shows ameasurable disruption of a biofilm. The effective amount will varydependent upon the stated function, the environment or element beingcontacted, the organism forming the biofilm or which is to be contacted,the state of development of the biofilm, among other conditions of theuse of the compound. It will be understood by one of ordinary skill inthe art, that for a given application, the effective amount can bedetermined by application of routine experimentation and without undueexperimentation by methods that are described herein or that are knownin the art.

The term “therapeutically effective amount” is used generically hereinto refer to the amount of a given compound or in case of a mixture thecombined amount of a mixture of components when administered to theindividual (including a human, or non-human animal) that provides ameasureable therapeutic effect for a listed disease, disorder orcondition to at least partially ameliorate a symptom of such disease,disorder or condition. The present invention provides methods oftreating disorders, diseases conditions and symptoms in a human ornon-human animal and particularly in a human, by administering to anindividual in need of treatment or prophylaxis, a therapeuticallyeffective amount of one or more compounds of this invention to theindividual in need thereof. The result of treatment can be partially orcompletely alleviating, inhibiting, preventing, ameliorating and/orrelieving the disorder, condition or one or more symptoms thereof. As isunderstood in the art, the therapeutically effective amount of a givencompound will depend at least in part upon, the mode of administration,any carrier or vehicle (e.g., solution, emulsion, etc.) employed, theextent of damage and the specific individual (human or non-human) towhom the compound is to be administered (age, weight, condition, sex,etc.). The dosage requirements needed to achieve the “therapeuticallyeffective amount” vary with the particular compositions employed, theroute of administration, the severity of the symptoms presented and theparticular subject being treated. Based on the results obtained instandard pharmacological test procedures, projected daily dosages ofactive compound can be determined as is understood in the art.

Administration is intended to encompass administration of a compound,pharmaceutically acceptable salt, solvate or ester thereof alone or in apharmaceutically acceptable carrier thereof or administration of aprodrug derivative or analog of a compound of this invention which willform an equivalent amount of the active compound or substance within thebody. An individual in need of treatment or prophylaxis includes thosewho have been diagnosed to have a given disorder or condition and tothose who are suspected, for example, as a consequence of the display ofcertain symptoms, of having such disorders or conditions.

Compounds of this invention can be employed in unit dosage form, e.g. astablets or capsules. In such form, the active compound or more typicallya pharmaceutical composition containing the active compound issub-divided in unit dose containing appropriate quantities of the activecompound; the unit dosage forms can be packaged compositions, forexample, packaged powders, vials, ampules, pre-filled syringes orsachets containing liquids. The unit dosage form can be, for example, acapsule or tablet itself, or it can be the appropriate number of anysuch compositions in package form.

The dosage can vary within wide limits and as is understood in the artwill have to be adjusted to the individual requirements in eachparticular case. By way of general guidance, the daily oral dosage canvary from about 0.01 mg to 1000 mg, 0.1 mg to 100 mg, or 10 mg to 500 mgper day of a compound of formulas herein or of the corresponding amountof a pharmaceutically acceptable salt thereof. The daily dose may beadministered as single dose or in divided doses and, in addition, theupper limit can also be exceeded when this is found to be indicated.

Any suitable form of administration can be employed in the methodherein. The compounds of this invention can, for example, beadministered in oral dosage forms including tablets, capsules, pills,powders, granules, elixirs, tinctures, suspensions, syrups andemulsions. Oral dosage forms may include sustained release or timedrelease formulations. The compounds of this invention may also beadministered topically, intravenously, intraperitoneally,subcutaneously, or intramuscularly, all using dosage forms well known tothose of ordinary skill in the pharmaceutical arts.

Compounds of this invention can also be administered in intranasal formby topical use of suitable intranasal vehicles. For intranasal orintrabronchial inhalation or insulation, the compounds of this inventionmay be formulated into an aqueous or partially aqueous solution, whichcan then be utilized in the form of an aerosol. Administration includesany form of administration that is known in the art and is intended toencompass administration in any appropriate dosage form and further isintended to encompass administration of a compound, alone or in apharmaceutically acceptable carrier. Pharmaceutical carriers areselected as is known in the art based on the chosen route ofadministration and standard pharmaceutical practice.

The compounds of this invention can also be administered to the eye,preferably as a topical ophthalmic formulation. The compounds of thisinvention can also be combined with a preservative and an appropriatevehicle such as mineral oil or liquid lanolin to provide an ophthalmicointment. The compounds of this invention may be administered rectallyor vaginally in the form of a conventional suppository. The compounds ofthis invention may also be administered transdermally through the use ofa transdermal patch containing the active compound and a carrier that isinert to the active compound, is non toxic to the skin, and allowsdelivery of the agent for systemic absorption into the blood stream viathe skin.

The compounds of the invention may be administered employing anocclusive device. A variety of occlusive devices can be used to releasean ingredient into the blood stream such as a semipermeable membranecovering a reservoir containing the active ingredient with or without acarrier, or a matrix containing the active ingredient. Other occlusivedevices are known in the literature.

Pharmaceutical compositions and medicaments of this invention compriseone or more compounds in combination with a pharmaceutically acceptablecarrier, excipient, or diluent. Such compositions and medicaments areprepared in accordance with acceptable pharmaceutical procedures, suchas, for example, those described in Remington's Pharmaceutical Sciences,17th edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton,Pa. (1985), which is incorporated herein by reference in its entirety.The invention also encompasses method for making a medicament employingone or more compounds of this invention which exhibit a therapeuticeffect.

Pharmaceutically acceptable carriers are those carriers that arecompatible with the other ingredients in the formulation and arebiologically acceptable. Carriers can be solid or liquid. Solid carrierscan include one or more substances that can also act as flavoringagents, lubricants, solubilizers, suspending agents, fillers, glidants,compression aids, binders, tablet-disintegrating agents, orencapsulating materials. Liquid carriers can be used in preparingsolutions, suspensions, emulsions, syrups and elixirs. The activeingredient can be dissolved or suspended in a pharmaceuticallyacceptable liquid carrier such as water (of appropriate purity, e.g.,pyrogen-free, sterile, etc.), an organic solvent, a mixture of both, ora pharmaceutically acceptable oil or fat. The liquid carrier can containother suitable pharmaceutical additives such as, for example,solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoringagents, suspending agents, thickening agents, colors, viscosityregulators, stabilizers or osmo-regulators. Compositions for oraladministration can be in either liquid or solid form.

Suitable solid carriers include, for example, calcium phosphate,magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin,cellulose, methyl cellulose, sodium carboxymethyl cellulose,polyvinylpyrrolidine, low melting waxes and ion exchange resins.Suitable examples of liquid carriers for oral and parenteraladministration include water of appropriate purity, aqueous solutions(particularly containing additives, e.g. cellulose derivatives, sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols e.g. glycols) and their derivatives,and oils. For parenteral administration, the carrier can also be an oilyester such as ethyl oleate and isopropyl myristate. Sterile liquidcarriers are used in sterile liquid form compositions for parenteraladministration. The liquid carrier for pressurized compositions can behalogenated hydrocarbon or other pharmaceutically acceptable propellant.Liquid pharmaceutical compositions that are sterile solutions orsuspensions can be administered by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministered intravenously. Compositions for oral administration can bein either liquid or solid form. The carrier can also be in the form ofcreams and ointments, pastes, and gels. The creams and ointments can beviscous liquid or semisolid emulsions of either the oil-in-water orwater-in-oil type. Pastes comprised of absorptive powders dispersed inpetroleum or hydrophilic petroleum containing the active ingredient canalso be suitable.

The term “pharmaceutically acceptable salts” refers to those salts whichretain the biological effectiveness and properties of the free bases orfree acids, which are not biologically or otherwise undesirable. Thesalts are formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, preferably hydrochloric acid, and organic acids such as aceticacid, propionic acid, glycolic acid, pyruvic acid, oxylic acid, maleicacid, malonic acid, succinic acid, fumaric acid, tartaric acid, citricacid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid,N-acetylcystein and the like.

In addition these salts may be prepared from addition of an inorganicbase or an organic base to the free acid. Salts derived from aninorganic base include, but are not limited to, the sodium, potassium,lithium, ammonium, calcium, magnesium salts and the like. Salts derivedfrom organic bases include, but are not limited to salts of primary,secondary, and tertiary amines, substituted amines including naturallyoccurring substituted amines, cyclic amines and basic ion exchangeresins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, lysine, arginine,N-ethylpiperidine, piperidine, polyimine resins and the like. Compoundsof formula I can also be present in the form of zwitterions.

Pharmaceutically acceptable salts comprise pharmaceutically-acceptableanions and/or cations. Pharmaceutically-acceptable cations include amongothers, alkali metal cations (e.g., Li+, Na+, K+), alkaline earth metalcations (e.g., Ca2+, Mg2+), non-toxic heavy metal cations and ammonium(NH4+) and substituted ammonium (N(R′)4+, where R′ is hydrogen, alkyl,or substituted alkyl, i.e., including, methyl, ethyl, or hydroxyethyl,specifically, trimethyl ammonium, triethyl ammonium, and triethanolammonium cations). Pharmaceutically-acceptable anions include amongother halides (e.g., Cl−, Br−), sulfate, acetates (e.g., acetate,trifluoroacetate), ascorbates, aspartates, benzoates, citrates, andlactate.

Compounds of the invention can have prodrug forms. Prodrugs of thecompounds of the invention are useful in the methods of this invention.Any compound that will be converted in vivo to provide a biologically,pharmaceutically or therapeutically active form of a compound of theinvention is a prodrug. Various examples and forms of prodrugs are wellknown in the art. Examples of prodrugs are found, inter alia, in Designof Prodrugs, edited by H. Bundgaard, (Elsevier, 1985), Methods inEnzymology, Vol. 42, at pp. 309-396, edited by K. Widder, et. al.(Academic Press, 1985); A Textbook of Drug Design and Development,edited by Krosgaard-Larsen and H. Bundgaard, Chapter 5, “Design andApplication of Prodrugs,” by H. Bundgaard, at pp. 113-191, 1991); H.Bundgaard, Advanced Drug Delivery Reviews, Vol. 8, p. 1-38 (1992); H.Bundgaard, et al., Journal of Pharmaceutical Sciences, Vol. 77, p. 285(1988); and Nogrady (1985) Medicinal Chemistry A Biochemical Approach,Oxford University Press, New York, pages 388-392).

The invention expressly includes pharmaceutically usable solvates ofcompounds according to formulas herein. The compounds of formula I canbe solvated, e.g. hydrated. The solvation can occur in the course of themanufacturing process or can take place, e.g. as a consequence ofhygroscopic properties of an initially anhydrous compound of formulasherein (hydration).

In specific embodiments herein, compounds 2, 13, 18E, 20E, 23E, 25E 30E,32, 33, 34, 35, 36, 37, 38, 39 are particularly useful for disruption ofbacterial quorum sensing and biofilm disruption, particularly in E.coli, V. fischeri and/or A. tumefaciens.

In specific embodiments herein, compounds 3, and 19E are particularlyuseful for activation of bacterial quorum sensing and biofilm formation,particularly in E. coli, V. fischeri and/or P. aeruginosa.

In specific embodiments herein, compounds 2, 13, 18E, 30E, 32, 33, 34,35, 36, 37 are particularly useful for disruption of bacterial quorumsensing and biofilm disruption in E. coli. In specific embodiments,compounds 1E, 18E, 20E, 22E, 23E, 25E, 26E, 27E, 28E, 30E, 33, 34, 36,38 and 39 are particularly useful for disruption of bacterial quorumsensing and biofilm disruption in V. fischeri. In specific embodimentsherein, compounds 1E, 26E, 27E, and 30E are particularly useful fordisruption of bacterial quorum sensing and biofilm formation,particularly in A. tumefaciens.

In specific embodiments herein, compounds 3, 14, 16, 17, 1E, 19E, 22E,26E, 27E, 28E, and 31 are particularly useful for activation ofbacterial quorum sensing and biofilm formation in E. coli. In specificembodiments herein compound 24E is particularly useful for activation ofbacterial quorum sensing and biofilm formation in A. tumefaciens. Inspecific embodiments herein compound 19E is particularly useful foractivation of bacterial quorum sensing and biofilm formation in V.fischeri. In specific embodiments herein compounds 3 and 1E areparticularly useful for activation of bacterial quorum sensing andbiofilm formation in P. aeruginosa.

In specific embodiments herein compounds of the formulas herein whichexhibit 20% or more inhibition in quorum sensing antagonism assays asdescribed in the examples herein are particularly useful for disruptionof bacterial quorum sensing and bacterial biofilm formation. In specificembodiments herein compounds of the formulas herein which exhibit 50% ormore inhibition in quorum sensing antagonism assays as described in theexamples herein are particularly useful for disruption of bacterialquorum sensing and bacterial biofilm formation. In specific embodimentsherein compounds of the formulas herein which exhibit 75% or moreinhibition in quorum sensing antagonism assays as described in theexamples herein are particularly useful for disruption of bacterialquorum sensing and bacterial biofilm formation.

In specific embodiments herein compounds of the formulas herein whichexhibit 20% or more activation in quorum sensing agonism assays asdescribed in the examples herein are particularly useful for activationof bacterial quorum sensing and bacterial biofilm formation. In specificembodiments herein compounds of the formulas herein which exhibit 50% ormore activation in quorum sensing agonism assays as described in theexamples herein are particularly useful for activation of bacterialquorum sensing and bacterial biofilm formation. In specific embodimentsherein compounds of the formulas herein which exhibit 75% or moreactivation in quorum sensing agonism assays as described in the examplesherein are particularly useful for activation of bacterial quorumsensing and bacterial biofilm formation.

Compounds of this invention are additionally useful as tools for use inresearch in the study of quorum sensing in bacteria.

Well-known methods for assessment of drugability can be used to furtherassess active compounds of the invention for application to giventherapeutic application. The term “drugability” relates topharmaceutical properties of a prospective drug for administration,distribution, metabolism and excretion. Drugability is assessed invarious ways in the art. For example, the “Lipinski Rule of 5” fordetermining drug-like characteristics in a molecule related to in vivoabsorption and permeability can be applied (C. A. Lipinski, F. Lombardo,B. W. Dominy, P. J. Feeney, Experimental and computational approaches toestimate solubility and permeability in drug discovery and developmentsettings, Adv. Drug Del. Rev., 2001, 46, 3-26 and Arup K. Ghose,Vellarkad N. Viswanadhan, and John J. Wendoloski, A Knowledge-BasedApproach in Designing Combinatorial or Medicinal Chemistry Libraries forDrug Discovery, J. Combin. Chem., 1999, 1, 55-68.) In general apreferred drug for oral administration exhibits no more than oneviolation of the following rules:

(1) Not more than 5 hydrogen bond donors (e.g., nitrogen or oxygen atomswith one or more hydrogens);

(2) Not more than 10 hydrogen bond acceptors (e.g., nitrogen or oxygenatoms);

(3) Molecular weight under 500 g/mol and more preferably between 160 and480; and

(4) log P less than 5 and more preferably between −0.4 to +5.6 and yetmore preferably −1<log P<2.

Compounds of this invention preferred for therapeutic applicationinclude those that do not violate one or more of 1-4 above.

Compounds of this invention preferred for therapeutic applicationinclude those having log P less than 5 and more preferably between −0.4to +5.6 and yet more preferably −1<log P<2.

The compounds of this invention may contain one or more chiral centers.Accordingly, this invention is intended to include racemic mixtures,diasteromers, enantiomers and mixture enriched in one or moresteroisomer. The scope of the invention as described and claimedencompasses the racemic forms of the compounds as well as the individualenantiomers and non-racemic mixtures thereof.

It is understood that this invention is not limited to the particularmethodology, protocols, cell lines, and reagents described, as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and equivalents thereof knownto those skilled in the art, and so forth. As well, the terms “a” (or“an”), “one or more” and “at least one” can be used interchangeablyherein.

As used herein, the term “treating” includes preventative as well asdisorder remittent treatment. As used herein, the terms “reducing”,“suppressing” and “inhibiting” have their commonly understood meaning oflessening or decreasing.

In certain embodiments, the present invention encompasses administeringthe compounds useful in the present invention to a patient or subject. A“patient” or “subject”, used equivalently herein, refers to an animal.In particular, an animal refers to a mammal, preferably a human. Thesubject either: (1) has a condition remediable or treatable byadministration of a compound of the invention; or (2) is susceptible toa condition that is preventable by administering a compound of thisinvention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference for the purpose of describing anddisclosing the chemicals, cell lines, vectors, animals, instruments,statistical analysis and methodologies which are reported in thepublications which might be used in connection with the invention.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

The invention includes compounds of formula I which exhibit activity asantagonist of quorum sensing in bacteria, particularly specific bacteriadisclosed herein. The invention also includes compounds of formula Iwhich exhibit activity as agonist of quorum sensing in bacteria,particularly specific bacteria disclosed herein.

In an embodiment, compounds of formula I have activity as an agonist orantagonist of native quorum sensing compounds. In an embodiment,compounds of formula I can be used to selectively adjust the virulence,biofilm production, or symbiotic behavior of a quorum sensing bacteria.In an embodiment, compounds of formula I can be administered to asubject to initiate an immune response towards a quorum sensingbacteria.

In an embodiment, certain compounds are preferred for selectivelyadjusting the virulence, biofilm production, or symbiotic behavior of aparticular species or strain of a particular species of quorum sensingbacteria. In an embodiment, preselected mixtures of L- and D-isomers ofcompounds of the present invention can be used to selectively adjust thevirulence, biofilm production, or symbiotic behavior of a particularspecies or strain of a particular species of quorum sensing bacteria.

In an embodiment, the compounds of the present invention are useful as acombinatorial library comprising a preselected mixture of two or morecompounds of the present invention. In an embodiment, the two or morecompounds can each be used to separately selectively adjust thevirulence, biofilm production, or symbiotic behavior of a particularspecies or strain of a particular species of quorum sensing bacteria.

When a group of substituents is disclosed herein, it is understood thatall individual members of those groups and all subgroups, including anyisomers and enantiomers of the group members, and classes of compoundsthat can be formed using the substituents are disclosed separately. Whena Markush group or other grouping is used herein, all individual membersof the group and all combinations and subcombinations possible of thegroup are intended to be individually included in the disclosure. When acompound is described herein such that a particular isomer or enantiomerof the compound is not specified, for example, in a formula or in achemical name, that description is intended to include each isomer andenantiomer of the compound described individually or in any combination.When an atom is described herein, including in a composition, anyisotope of such atom is intended to be included. Specific names ofcompounds are intended to be exemplary, as it is known that one ofordinary skill in the art can name the same compounds differently. Everyformulation or combination of components described or exemplified hereincan be used to practice the invention, unless otherwise stated. Whenevera range is given in the specification, for example, a temperature range,a time range, or a composition range, all intermediate ranges andsubranges, as well as all individual values included in the ranges givenare intended to be included in the disclosure.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art, insome cases as of their filing date, and it is intended that thisinformation can be employed herein, if needed, to exclude (for example,to disclaim) specific embodiments that are in the prior art. Forexample, when a compound is claimed, it should be understood thatcompounds known in the prior art, including certain compounds disclosedin the references disclosed herein (particularly in referenced patentdocuments), are not intended to be included in the claim.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention. It will be apparent to oneof ordinary skill in the art that methods, devices, device elements,materials, procedures and techniques other than those specificallydescribed herein can be applied to the practice of the invention asbroadly disclosed herein without resort to undue experimentation. Allart-known functional equivalents of methods, devices, device elements,materials, procedures and techniques described herein are intended to beencompassed by this invention. Whenever a range is disclosed, allsubranges and individual values are intended to be encompassed. Thisinvention is not to be limited by the embodiments disclosed, includingany shown in the drawings or exemplified in the specification, which aregiven by way of example or illustration and not of limitation.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments, exemplary embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims. The specific embodiments provided herein are examplesof useful embodiments of the present invention and it will be apparentto one skilled in the art that the present invention may be carried outusing a large number of variations of the devices, device components,methods steps set forth in the present description. As will be obviousto one of skill in the art, methods and devices useful for the presentmethods can include a large number of optional composition andprocessing elements and steps.

Many of the molecules disclosed herein contain one or more ionizablegroups [groups from which a proton can be removed (e.g., —COOH) or added(e.g., amines) or which can be quaternized (e.g., amines)]. All possibleionic forms of such molecules and salts thereof are intended to beincluded individually in the disclosure herein. With regard to salts ofthe compounds herein, one of ordinary skill in the art can select fromamong a wide variety of available counterions those that are appropriatefor preparation of salts of this invention for a given application. Inspecific applications, the selection of a given anion or cation forpreparation of a salt may result in increased or decreased solubility ofthat salt.

Every formulation or combination of components described or exemplifiedherein can be used to practice the invention, unless otherwise stated.

Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition or concentrationrange, all intermediate ranges and subranges, as well as all individualvalues included in the ranges given are intended to be included in thedisclosure. It will be understood that any subranges or individualvalues in a range or subrange that are included in the descriptionherein can be excluded from the claims herein.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when composition ofmatter are claimed, it should be understood that compounds known andavailable in the art prior to Applicant's invention, including compoundsfor which an enabling disclosure is provided in the references citedherein, are not intended to be included in the composition of matterclaims herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be replaced with either of the other terms. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein.

One of ordinary skill in the art will appreciate that startingmaterials, biological materials, reagents, synthetic methods,purification methods, analytical methods, assay methods, and biologicalmethods other than those specifically exemplified can be employed in thepractice of the invention without resort to undue experimentation. Allart-known functional equivalents, of any such materials and methods areintended to be included in this invention. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention is further illustrated by the following non-limitingexamples.

THE EXAMPLES Example 1

Heterocycles and carbocycles were chosen as head groups for a library(FIG. 4) in order to probe the orientation and electronics necessary fora positive binding interaction with Trp60, shown to be an importantresidue in the N-terminal domain of the LasR crystal structure. Fluorinewas chosen as a lactone carbonyl mimic due to its ability to accepthydrogen bonds. Multiple fluorine aromatic substitutions were examinedto determine if Trp60 could hydrogen bond to multiple atoms given thecorrect spatial orientation. Non-hydrogen bonding oxygen-containingmoieties were chosen to examine the effects of non-hydrogen bondingelectrostatic interactions. The library also contained carbocycles toexplore the necessity of the Trp60 binding interaction. A thiolactoneanalog shown to be active in previous experiments was chosen to serve asa control compound for our bacterial strains. [Passador, L.; Tucker, K.D.; Guertin, K. R.; Journet, M. P.; Kende, A. S.; Iglewski, B. H.,Functional analysis of the Pseudomonas aeruginosa Autoinducer PAI. J.Bacteriol. 1996, 178, 5995-6000.] The glycine ethyl ester and thealanine methyl ester were chosen to explore the effects of variations onsynthetic ring-opened forms of the lactone unavailable to nature.

Based upon this design strategy, a 17 member non-lactone based library(FIG. 4) was synthesized using solution-phase chemistry. To facilitatethe ease of synthesis, a Meldrum's Acid derivative was used as a commonintermediate. Reacting Meldrum's Acid with decanoyl chloride affordedthe Meldrum's Acid derivative, which was refluxed with the desiredamines to form the initial library (Scheme 1).

Scheme 1 is a general synthetic method for producing 3-oxo-dodecanoyalderivatives of the natural autoinducer for P. aeruginosa. DMAP=dimethylamino pyridine. TEA=triethyl amine. R can, for example, be aunsubstituted or substituted heterocycle or carbocycle:

This method can be employed for synthesis of various compounds herein bychoice of starting materials and routine adaptation of methods disclosedherein or of methods that are well-known in the art. This method can beused for synthesis of compounds, where R is various substituted andunsubstituted heterocyclic rings, in particular, where R is a ringsubstituted thiolactone group. Appropriate starting materials for makingring-substituted compounds of this inventions are readily availableeither form commercial sources or by known synthetic methods. Additionalreferences which provide details useful in the synthesis of thiolactonesof this invention include among others U.S. Pat. Nos. 3,840,534 and3,926,965 and Krasncv et al. (1999) Russian J. Org. Chem. 35(4):572-577.

The initial library was tested for LasR agonistic and antagonisticactivity in two strains: Escherichia coli DH5α (pJN105L+pSC11) [Lee, J.H.; Lequette, Y.; Greenberg, E. P., Activity of purified QscR, aPseudomonas aeruginosa orphan quorum-sensing transcription factor. Mol.Microbiol. 2006, 59 (2), 602-609] and P. aeruginosa PA01 MW1 (pUM15)[Muh, U.; Schuster, M.; Heim, R.; Singh, A.; Olson, E.; Greenberg, E.P., Novel Pseudomonas aeruginosa Quorum-Sensing Inhibitors Identified inan Ultra-High-Throughput Screen. Antimicrob. Agents Chemother. 2006, 50,3674-3679] (FIGS. 5A and 5B). Both strains allow for syntheticautoinducer mimic evaluation and contain a reporter gene that allows fora quantitative readout of QS activity. DH5α (pJN105L+pSC11) is aheterologous β-galactosidase E. coli reporter strain containing aplasmid for the P. aeruginosa LasR gene. The PA01 MW1 (pUM15) strainuses the natural P. aeruginosa background containing a LasI deletion andthe gene for yellow fluorescent protein (YFP) under the control of theLasI promoter to evaluate the LasI/R activity. Since the PA01 MW1(pUM15) strain evaluates LasR in the natural P. aeruginosa background,in contrast to the heterologous E. coli strain, an improved idea of theinterplay between the isolated LasI/R system and the combination ofLasI/R with other QS subsystems such as QscR can be determined.Furthermore, additional nuances of the natural system such as compoundpermeability are incorporated in assays using the PA01 MW1 (pUM15)strain.

The initial library was also tested in Vibrio fischeri ESI 114 (Δ-LuxI)[Lupp, C.; Urbanowski, M.; Greenberg, E. P.; Ruby, E. G., The Vibriofischeri quorum-sensing systems ain and lux sequentially induceluminescence gene expression and are important for persistence in thesquid host. Mol. Microbiol. 2003, 50 (1), 319-331] and Agrobacteriumtumefaciens WCF (pCF372). [Zhu, J.; Beaber, J. W.; More, M. I.; Fuqua,C.; Eberhard, A.; Winans, S. C., Analogs of the autoinducer3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraRprotein of Agrobacterium tumefaciens. J. Bacteriol. 1998, 180 (20),5398-5405.] However, activities were low to modest in these species,except for compound 1, which is a good antagonist in both strains. Thegeneral lack of activity in the V. fischeri and A. tumefaciens strainsis to be expected considering that the library was designed for the P.aeruginosa LasR protein and reinforces previous work demonstrating thatthe length of the acyl tail is highly species dependent. [Geske, G. D.;O'Neill, J. C.; Miller, D. M.; Mattmann, M. E.; Blackwell, H. E.,Modulation of Bacterial Quorum Sensing: Systematic Evaluation ofN-Acylated Homoserine Lactones in Multiple Species and New Insights intoTheir Mechanism of Action. J. Am. Chem. Soc. 2007, 129, 13613-13625.]

This set of screening data provides several noteworthy discoveries.First, the thiolactone derivative of the P. aeruginosa natural ligand(1) is highly active in all strains tested—either as an agonist in thestrains examining LasR activity [94% agonist in DH5α (pJN105L+pSC11) and88% agonist in PA01 MW1 (pUM15)] or as an antagonist in the V. fischeri(LuxR; 92% inhibition) and A. tumefaciens (TraR; 65% inhibition)strains. The high degree of activity across these four strains suggeststhat the sulfur substitution in the lactone ring does not sufficientlyalter the binding of 1 from the natural ligand and suggests that theelectronics at that position are not critical for binding. Second, thecyclopentyl amine derivative (3) is an agonist in both strains testingfor LasR activity and a modest antagonist in the V. fischeri strain.This is remarkable because the cyclopentyl amine head group lacksfunctionality for hydrogen bond acceptance, which has been proposed ascritical for neutral ligand binding based on the LasR crystal structure.This suggests that either the lactone carbonyl's hydrogen bonds are notas crucial as originally thought, or that 3 binds in an alternativemanner. Third, compound 2 is of interest for its antagonism capabilitiesin both strains evaluating the LasI/R system, even though there are nohydrogen bond acceptor substitutions, further questioning the proposedcritical nature of the lactone carbonyl. Fourth, compound 14 providessome insight into the differences between the isolated LasI/R system inE. coli and the LasI/R system in the natural P. aeruginosa background.LasR appears to be strongly agonized by 14 in the E. coli strain, whileassays using the natural P. aeruginosa background show slight antagonismrather than an agonistic effect. Compound 14 represented an excellentcandidate for further testing in additional heterologous strainscontaining isolated QS subsystems like QscR and RhII/R.

Additional comparative data for several compounds of FIG. 4 is providedin Table 1

TABLE 1 E. coli DH5α (pJN105L + P. aeruginosa V. fischeri A. TumefacienspSC11) PA01 MW1 ESI 114 (Δ-Luxl) WCF (pCF372) Compd Antagonist AgonistAntagonist Agonist Antagonist Agonist Antagonist Agonist 2 53.7 — 28.7 —— — — — 3 — 83.5 — 54.2 89.4 — — — 7 38.8 — — — — 3.1 — — 10 — — 25.5 —— 3.3 — — 12 — — −6.6  4.0 34.9 3.0 −22.0 — 13 41.0 — 16.8 — 43.8 1.7 —— 14 −67.7 73.0 37.8 — 14.6 — — — Agonist results are reported as apercent of activation compared to the positive control. Antagonistresults are reported as a percent inhibition compared to a positivecontrol. Negative values indicate agonist properties detected in anantagonist assay.

Example 2 Focused Libraries—Racemic Thiolactone Library

Based upon the results of the initial library screen, focused librariesaround the most active leads (1, 2, 3, 16 from FIG. 4) were developed.In these libraries, the identified head group remained identical whilethe 3-oxo-C12 acyl tail was replaced with mimics previously shown to beactive in AHL libraries. [Geske, G. D.; O'Neill, J. C.; Miller, D. M.;Mattmann, M. E.; Blackwell, H. E., Modulation of Bacterial QuorumSensing: Systematic Evaluation of N-Acylated Homoserine Lactones inMultiple Species and New Insights into Their Mechanism of Action. J. Am.Chem. Soc. 2007, 129, 13613-13625.]

The first focused library (FIG. 6) was a racemic homoserine thiolactonelibrary, synthesized from 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) couplings between homoserine thiolactone and theappropriate carboxylic acid (Scheme 2).

Scheme 2 provides EDC coupling synthesis to make the racemic thiolactonelibrary illustrated in FIG. 6.

This method can be employed for synthesis of various compounds herein bychoice of starting materials and routine adaptation of methods disclosedherein or of methods that are well-known in the art. This method can beused for synthesis of compounds, where R is various substituted andunsubstituted heterocyclic rings, in particular, where R is a ringsubstituted thiolactone group. Appropriate starting materials for makingring-substituted compounds of this inventions are readily availableeither form commercial sources or by known synthetic methods. Additionalreferences which provide details useful in the synthesis of thiolactonesof this invention include among others U.S. Pat. Nos. 3,840,534 and3,926,965 and Krasncv et al. (1999) Russian J. Org. Chem. 35(4):572-577.

The racemic thiolactone library was tested in the same LasR reporterstrains as the initial library (FIGS. 7A and 7B). Differences betweenthe isolated LasR reporter system and LasR reporter system in thenatural P. aeruginosa background have been uncovered in this secondgeneration library. All of the library members were active in theheterologous LasI/R system while inactive in the intact P. aeruginosa QSsystem. One possibility is that library members are regulating multiplecompeting QS pathways, resulting in net inactivity in the naturalbackground. For this reason it is crucial to examine the LasI/R systemin the natural P. aeruginosa background where additional QS subsystemsare also present and not simply as an isolated system in the E. colibackground. Differences in cell permeability, especially since P.aeruginosa is known to be less permeable than E. coli, could alsoaccount for the discrepancy in activity between the two strains.

Example 3 Focused Libraries—Enantiopure Thiolactone Library

After finding several active compounds in the racemic thiolactonelibrary in the heterologous E. coli LasI/R strain and the V. Fischeristrain, a third generation library was designed containing theenantiopure thiolactone along with additional acyl chain mimics tofurther explore the structure-activity relationship of the thiolactonehead group (FIG. 8). The L enantiomer of the thiolactone was chosenbased on previous studies that found the L enantiomer of P. aeruginosa'snatural autoinducer to be active and the D enantiomer to be inactive.[Geske, G. D.; O'Neill, J. C.; Miller, D. M.; Mattmann, M. E.;Blackwell, H. E., Modulation of Bacterial Quorum Sensing: SystematicEvaluation of N-Acylated Homoserine Lactones in Multiple Species and NewInsights into Their Mechanism of Action. J. Am. Chem. Soc. 2007, 129,13613-13625; Glansdorp, F. G.; Thomas, G. L.; Lee, J. K.; Dutton, J. M.;Salmond, G. P. C.; Welch, M.; Spring, D. R., Synthesis and stability ofsmall molecule probes for Pseudomonas aeruginosa quorum sensingmodulation. Org. Biomol. Chem. 2004, 2, 3329-3336.]

FIG. 8 provides structures (with reference numbers) of compounds of theenantiopure thiolactone library synthesized from a combination ofMeldrum's acid precursors and EDC couplings.

The third generation thiolactone compounds were similarly tested inbacterial assays beside the racemic version, if synthesized, todetermine the effect of stereochemistry on activity (FIGS. 9A-9H). FIGS.9A-9H provide a comparison between racemic and enantiopure thiolactoneanalogs. All synthetic ligands were tested at 10 μM using standardmethods described in FIGS. 5A and 5B. Compounds 24-30 of the enantiopurelibrary were not compared to a racemic counterpart. If stereochemistryplayed a large role in binding, the enantiopure compounds were expectedto have approximately twice the activity of the racemic compounds whenscreened at 10 μM total synthetic ligand in each case. In both strainstesting for LasR activity, it appeared, however, that stereochemistrywas not important for activity of the non-native thiolactones incontrast to the lactones. Without wishing to be bound by any particulartheory, we presently believe that binding is likely less specific forthe thiolactones compared to the natural lactone ligand, where it isknown that the L enantiomer is far more active than the D enantiomer.

Dose response analysis for the active enantiopure thiolactone compoundswas conducted to quantify the activity of the synthetic ligands (Table2). The activity of the thiolactone head group alone became evidentthrough compound 21E in the heterologous LasR strain, whose AHL analoguewas found to have little activity. [Geske, G. D.; O'Neill, J. C.;Miller, D. M.; Mattmann, M. E.; Blackwell, H. E., Modulation ofBacterial Quorum Sensing: Systematic Evaluation of N-Acylated HomoserineLactones in Multiple Species and New Insights into Their Mechanism ofAction. J. Am. Chem. Soc. 2007, 129, 13613-13625.] Based on the resultsof the enantiopure thiolactone dose response data, it appears thatrelatively long or electron withdrawing side chains are excellentantagonists of LasR isolated in the E. coli background. However, naturalligand mimics 1E and 25E are strong agonists for the heterologous LasRsystem. All of the active compounds in PA01 MW1 (PUM15) (P. aeruginosanatural background) were also active in E. coli DH5α (pJN105L+pSC11).However, a significant number of the compounds active in the E. coliDH5α (pJN105L+pSC11) strain were not active in the P. aeruginosa PA01MW1 (PUM15) strain. These findings corroborate our hypothesis that whenLasR is evaluated in the natural P. aeruginosa background a muted effectis may be seen due to the effects of the other QS systems present inintact P. aeruginosa, such as QscR and the PQS system. A variety ofother effects could be contributing to the differences seen between thetwo strains, including differences in cell permeability.

TABLE 2 Table 2. The IC₅₀ and EC₅₀ values for the most activeenantiopure thiolactone library members. DH5α (pJN105L + PA01 MW1 ESI114 (Δ- pSC11) (pUM15) Luxl) WCF (pCF372 E. Coli P. aeruginosa V.fischeri A. tumefaciens IC₅₀ EC₅₀ IC₅₀ EC₅₀ IC₅₀ EC₅₀ IC₅₀ EC₅₀ Comp. #(μM) (μM) (μM) (μM) (μM) (μM) (μM) (μM)  1E 0.092 3.2 0.45 1.8 18E 0.400.77 19E 4.1 11 20E 7.2 21E 2.5 22E 1.8 23E 2.9 0.35 24E 0.35 20 25E 1.921 26E 0.14 0.13 2.8 27E 0.79 0.31 10 28E 1.1 0.84 30E 0.13 13 3.2

Activity differences between the two strains evaluating LasR raisequestions about the integrity and degradation of the ligands since theincubation time in the assay for the heterologous strain is shorter thanfor the native P. aeruginosa strain. It is well known that thehomoserine lactone ring, used by all of the bacterial species ofinterest as their autoinducer head group, is prone to hydrolysis at pH 7and above. [Eberhard, A.; Widrig, C. A.; MaBath, P.; Schineller, J. B.,Analogs of the autoinducer of bioluminescence in Vibrio fischeri. Arch.Microbiol. 1986, 146, 35-40; Schaefer, A. L.; Hanzelka, B. L.; Eberhard,A.; Greenberg, E. P., Quorum sensing in Vibrio fischeri: Probingautoinducer-LuxR interactions with autoinducer analogs. J. Bacteriol.1996, 178, 2897-2901; Byers, J. T.; C., L.; Salmond, G. P. C.; Welch,M., Nonenzymatic turnover of an Erwinia carotovora quorum sensingsignaling molecule. J. Bacteriol. 2002, 184, 1163-1171.] Previousliterature has indicated that the P. aeruginosa natural autoinducer hasa half-life of approximately two days in growth media at 37° C., whileshorter chain AHLs degrade in even shorter periods of time. [Glansdorp,F. G.; Thomas, G. L.; Lee, J. K.; Dutton, J. M.; Salmond, G. P. C.;Welch, M.; Spring, D. R., Synthesis and stability of small moleculeprobes for Pseudomonas aeruginosa quorum sensing modulation. Org.Biomol. Chem. 2004, 2, 3329-3336; Yates, E. A.; Philipp, B.; Buckley,C.; Atkinson, S.; Chhabra, S. R.; Sockett, R. E.; Goldner, M.; Dessaux,Y.; Camara, M.; Smith, H.; Williams, P., N-Acylhomoserine lactonesundergo lactonolysis in a pH-, temperature-, and acyl chain lengthdependent manner during growth of Yersinia pseudotuberculosis andPseudomonas aeruginosa. Infect. Immun. 2002, 70, 5635-5646]

Finding QS antagonists and agonists that are more hydrolytically stableare of considerable interest, since molecules that hydrolyze rapidly arenot ideal therapeutic agents or biological probes. While many of thecompounds synthesized in the initial library are non-hydrolyzable, thethiolactone derivative of the natural ligand (1E) is hydrolyzable.However, the differences in activities between the thiolactonederivatives and the natural lactone derivatives make the thiolactonederivatives both worthwhile to pursue as a target and for furtherhalf-life experiments.

Table 3 provides a summary of data for the antagonism assay forcompounds tested having thiolactone head groups against selectedbacteria. Compounds exhibiting 50% or higher inhibition in assays withEscherichia coli and Agrobacterium tumefaciens and those exhibiting 20%or higher inhibition with Vibrio fischeri are preferred for applicationsfor disrupting bacterial quorum sensing, particularly in Escherichiacoli, Agrobacterium tumefaciens and Vibrio fischeri strains, and forinhibiting and/or disrupting biofilm formation, particularly inEscherichia coli, Agrobacterium tumefaciens and Vibrio fischeri strains.

TABLE 3 Antagonism Assay Data Thiolactone Libraries E. coli V. fischeriA. tumefaciens Comp # Inhib % Comp # Inhib % Comp # Inhib % 18 80  1 9926E 99 27E 78 24E 99 30E 93 18E 68 23 98  1 92 21 65 18 97 27E 78 26E 6428E 93 1E 51 21E 61 1E 91 19 33 28E 59 18E 91 19E 30 23 56 23E 85 23E 2622E 54 26E 80 18E 25 20 51 27E 78 21E 13 20E 48 22E 75 23 9 23E 45 20 7025E 6 29E 17 22 68 21 −22 22 16 30E 62 20E −24 19E −3 20E 59 22E −26 19−13 25E 57 18 −60 24E −30 29E 42 29E −70  1 −40 21 34 28E −78  1E −6119E 30 22 −80 25E −93 21E 30 24E −89 30E −119 19 19 20 −107

Table 4 provides a summary of data for the agonism assay for compoundstested having thiolactone head groups with certain bacteria. Compoundsexhibiting 50% or higher inhibition in assays with Escherichia coli andP. aeruginosa are preferred for applications for activating bacterialquorum sensing, particularly in Escherichia coli, and P. aeruginosastrains and for activating biofilm formation therein.

TABLE 4 Agonism Assay Data for Thiolactones E. coli P. aeruginosa Comp #Act % Comp# Act %  1E 102  1E 127 1 94 1 88 26E 85 30E 76 19   82 25E 4219E 81 29E 22 22E 72 28E 20 23   9 21E 17 18E 8 22   10 27E 8 18   8 30E6 20   7 22   4 18E 5 25E 4 19   5 29E 4 20E 4 21E 3 19E 3 18   2 22E 321   2 23   3 20   1 24E 3 28E 1 21   2 20E 0 23E 1 23E 0 26E 0 24E 027E 0

Example 4 Comparison of Functional Half-Lives of Autoinducers

A biologically based assay was developed to determine the functionalhalf-life of the P. aeruginosa natural ligand, OdDHL, and thecorresponding thiolactone analog (1E). This assay does not directlymeasure hydrolysis, but rather the ability of the degraded ligand tocause a QS response. However, previous experiments have shown that thehydrolysis half-life for the P. aeruginosa natural ligand, OdDHL, isapproximately two days, while racemization of the chiral center wasfound to be less than 5% over the course of a week. [Glansdorp, F. G.;Thomas, G. L.; Lee, J. K.; Dutton, J. M.; Salmond, G. P. C.; Welch, M.;Spring, D. R., Synthesis and stability of small molecule probes forPseudomonas aeruginosa quorum sensing modulation. Org. Biomol. Chem.2004, 2, 3329-3336.] This data suggests that most ligand degradation isdue to hydrolysis and not epimerization. In these cases the results weredetermined by NMR experiments conducted in deuterated buffers. Due toproblems with the water solubility of OdDHL, a 50% solution of DMSO wasused. Unfortunately, the use of high levels of DMSO reduces thebiological relevance of the assay because large concentrations of DMSOcannot be tolerated by biological systems. Furthermore, the requiredconcentrations of ligand are lower in a biologically based functionalassay than in an NMR experiment because YFP production is much moresensitive than NMR, which requires relatively high concentrations.

In this assay, media, ligand, and antibiotics are prepared inTeflon-capped vials and allowed to incubate at 37° C. for predeterminedtimes. P. aeruginosa cells from the strain PA01 MW1 (pUM15) werecultured overnight. These cells were then pelleted and washed with LBcontaining 50 mM MOPS. After washing, the cells were resuspended in aminimal amount of media containing antibiotics and were added to a 96well plate containing the media, natural ligand, and antibioticpreviously prepared and incubated for specific, predetermined times. Atthis point, the optical density at 600 nm of the cells was comparable tothe optical density after subculturing the cells during a traditionalassay. The 96 well plate was incubated for 8 hours and then analyzed foroptical density and YFP fluorescence. Bacteria were cultured, pelleted,and washed before addition to the assay plate. A traditional PA01 MW1assay is completed to analyze for ligand degradation. Fluorescence wasnormalized to cell density and time points were analyzed as a percentageof the ability of the freshly prepared natural ligand to agonize the P.aeruginosa system. Since we predict that the ligand degradation is aproduct of hydrolysis, we assumed a pseudo first order rate and plottedthe natural log of the agonism as a percent of the fresh natural ligandversus time. The slope of the graph can be used to determine thehalf-life of the ligand according to the formula t½=ln(2)/slope (FIGS.10A and 10B).

We found the half-life for the OdDHL natural ligand to be 48.2 hours.This value corresponds closely with the previously found hydrolysishalf-life of approximately two days. [Glansdorp, F. G.; Thomas, G. L.;Lee, J. K.; Dutton, J. M.; Salmond, G. P. C.; Welch, M.; Spring, D. R.,Synthesis and stability of small molecule probes for Pseudomonasaeruginosa quorum sensing modulation. Org. Biomol. Chem. 2004, 2,3329-3336.]

A similar analysis found the half-life of the thiolactone analog of theOdDHL P. aeruginosa natural ligand (1E) to be 82.3 hours. In the case ofboth the natural ligand and the thiolactone analog, the half-life of thecompounds are sufficiently long so that standard in vitro assays on thetime scale of 8 hours or less are testing the ligand in its native form.This is important because previous work has shown that the ring openform of the natural ligand is inactive. [Yates, E. A.; Philipp, B.;Buckley, C.; Atkinson, S.; Chhabra, S. R.; Sockett, R. E.; Goldner, M.;Dessaux, Y.; Camara, M.; Smith, H.; Williams, P., N-Acylhomoserinelactones undergo lactonolysis in a pH-, temperature-, and acyl chainlength dependent manner during growth of Yersinia pseudotuberculosis andPseudomonas aeruginosa. Infect. Immun. 2002, 70, 5635-5646; Kapadnis, P.B.; Hall, E.; Ramstedt, M.; Galloway, W. R. J. D.; Welch, M.; Spring, D.R., Towards quorum-quenching catalytic antibodies. Chem. Commun. 2009,(5), 538-540.]

It is interesting to note that the half-life of 1E is slightly less thandouble the half-life of OdDHL, the natural ligand. This is particularlyintriguing because one would expect the sulfur analog to have a fasterhydrolysis rate from an electronics argument. Our current hypothesis isthat although compound 1E is able to ring open faster than OdDHL, 1E isalso able to recyclize at a faster rate than the natural ligand.Previous analysis of lactone hydrolysis has shown that once ring opened,the lactone does not reclose in appreciable quantities until under pH 2due to differences in the mechanisms for ring opening and closing.[Yates, E. A.; Philipp, B.; Buckley, C.; Atkinson, S.; Chhabra, S. R.;Sockett, R. E.; Goldner, M.; Dessaux, Y.; Camara, M.; Smith, H.;Williams, P., N-Acylhomoserine lactones undergo lactonolysis in a pH-,temperature-, and acyl chain length dependent manner during growth ofYersinia pseudotuberculosis and Pseudomonas aeruginosa. Infect. Immun.2002, 70, 5635-5646.} In order for the lactone ring to close, the pHmust first approach the pKa of the carboxyl group so that significantamounts of the acid as opposed to the acid salt are present. Thedifferences in ring opening and closing mechanisms may cause the naturalligand to take longer to ring open, but remain ring opened, while thesulfur analog would ring open faster and close back up again so that itwould have a longer hydrolysis half-life than the natural ligand.Conversely, the sulfur's increased nucleophilicity may be able to holdthe lactone ring together in aqueous solution better than thecorresponding oxygen.

Example 5 Focused Libraries—Non-Hydrolyzable Head Groups

While many of the natural ligands for QS systems contain a lactone ring,it would be advantageous to find QS modulators that are not prone tohydrolysis or degradation. To this end focused libraries based onnon-hydrolyzable head groups screened in the initial library weredesigned, synthesized, and screened. Head groups based upon glycineethyl ester (16), cyclopentyl amine (3), and aniline (2) were chosen asparticularly interesting non-hydrolyzable head groups based on activityin the initial library screens.

The glycine ethyl ester head group is particularly interesting becausethe stereochemistry has been removed from the head group. The glycineethyl ester head group is derived from the lactone ring when adisconnection is made between the carbons 2 and 3 in the lactone ring.(Scheme 3).

While the compounds of this library (FIG. 11) are non-natural analogs ofthe lactone ring, it is interesting that some activity can be observed.One characteristic of this library is that the compounds appear to becooperative agonists because many of the library members show heightenedactivity in antagonistic assays and minimal activity in agonistic assays(FIGS. 12A and 12B). Compound 34 showed excellent antagonistic activityin V. fischeri. While most library members do not seem to fit astraditional agonists or antagonists, further analysis could yieldimportant information about alternative binding sites or methods, orinformation about dimerization requirements.

Cyclopentyl amine and aniline were used to synthesize libraries toexplore the activity of ligands with a lack of hydrogen bondingcapabilities on the head group (FIG. 13 (cyclopentyl amine libraryHG=cyclopentyl), FIG. 14 (analine library, HG=phenyl)). Agonism andantagonism assays, performed as described above, are illustrated inFIGS. 15A and 15B, respectively. While these carbocycles were activewhen appended with the 3-oxo dodecanoyal containing acyl chain, onlymoderate activities were observed when paired with acyl tail mimics.These studies show that viable agonists and antagonists can be foundeither by altering the head group of the natural ligand or by creatingacyl tail mimics. However, when both the head group and the acyl tailare modified in the same molecule, the molecule doesn't always combinethe activities of the two initial modifications. In fact, the dualmodifications are frequently deleterious to the activity of themolecule.

Pursuing QS modulators that are either non-hydrolyzable or hydrolyzeslowly allows for new biological probes or therapeutics. It is importantfor therapeutics to remain biologically active for extended periods oftime yet be cleared from the body in a time dependent manner. Compoundslike the thiolactone derivatives of this invention may serve asexcellent therapeutics because they are active for longer periods oftime than the natural lactone analogs, yet do lose activity in a timedependent fashion.

We claim:
 1. A compound of formula:A—CO—CH₂—CO—NH—[CH₂]_(p2)-HG or a pharmaceutically acceptable salt orester thereof where: p2 is 1 or 2; A is an unsubstituted alkyl oralkenyl group having 4-10 carbon atoms; and HG is a phenyl groupsubstituted with one or more substituents selected from the groupconsisting of halogen, nitro, hydroxyl, nitrile, azide, —R, or —ORwherein R is an alkyl group having 1-6 carbon atoms.
 2. The compound ofclaim 1 wherein HG is a phenyl group substituted with one or moresubstituents selected from the group consisting of fluorine, nitro,nitrile, azide, —R, or —OR wherein R is an alkyl group having 1-6 carbonatoms.
 3. The compound of claim 1 wherein p2 is
 1. 4. The compound ofclaim 1 wherein A is an unsubstituted alkyl group having 4-10 carbonatoms.
 5. A compound represented by formula:

or a pharmaceutically acceptable salt or ester thereof wherein HG is aphenyl group optional substituted with one or more non-hydrogensubstituents selected from the group consisting of halogen, nitro,azide, alkyl having 1-3 carbon atoms, haloalkyl having 1-3 carbon atoms,alkoxy having 1-3 carbon atoms, and p2 is 1 or
 2. 6. The compound ofclaim 5 represented by formula:


7. The compound of claim 5 which has formula:


8. A pharmaceutical composition comprising one or more compounds ofclaim 1 and a pharmaceutically acceptable carrier.
 9. The composition ofclaim 8 further comprising an antimicrobial compound, an antibacterialcompound, an antiviral agent or an antibiotic.
 10. A method forinhibiting of quorum sensing in bacteria which comprising the step ofcontacting the quorum sensing bacteria with an amount of a quorumsensing compound of claim 1 effective for inhibiting of quorum sensing.11. A method for disruption of bacterial biofilm formation whichcomprises the step of contacting the biofilm or an environmentcontaining the biofilm with one or more compounds of claim 1.